Kjeldahl Method for Nitrogen Estimation

Kjeldahl Method

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.

Method Description:

Mechanism

The Kjeldahl method was developed in 1883 by a brewer called Johann Kjeldahl. A food is digested with a strong acid so that it releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements. It is usually considered to be the standard method of determining protein concentration. Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications, however, this is only an average value, and each protein has a different conversion factor depending on its amino-acid composition. The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration.

Digestion

The food sample to be analyzed is weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction). Digestion converts any nitrogen in the food (other than that which is in the form of nitrates or nitrites) into ammonia, and other organic matter to C0₂ and H₂0. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH₄⁺) which binds to the sulfate ion (SO₄^2-) and thus remains in solution:

Digestion is accomplished by boiling a homogeneous sample in concentrated sulfuric acid. The end result is an ammonium sulfate solution. The general equation for the digestion of an organic sample is shown below:

Organic N + H2SO4    →

(NH4)SO4 + H2O + CO4 + other sample matrix byproducts

Distillation: Excess base is added to the digestion product to convert NH₄ to NH₃as indicated in the following equation. The NH₃ is recovered by distilling the reaction product.

ammonium sulfate	heat	ammonia gas
(NH4)2SO4 + 2NaOH	→	2NH3 + Na2SO4 + 2H2O

TitrationTitration 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	standard sulfuric acid acid		excess ammonium sulfate	sulfuric acid
2NH3 +	2H2SO4	→	(NH4)2SO4 +	H2SO4
(no color change)

ammonia sulfate	measured excess acid	measured sodium hydroxide		ammonium sulfate
(NH4)2SO4 +	H2SO4 +	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	boric acid		ammonium- borate complex	excess boric acid
NH3 +	H3BO3	→	NH4 + H2BO-3 +	H3BO3
(color change occurs)

The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia collects, the color of the receiving solutions changes.

ammonium- borate complex		sulfuric acid		ammonium sulfate		boric acid
2NH4	+	H2BO-3	+	H2SO4 (NH4)2SO4	+	2H3BO3
(color change occurs in reverse)

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.

Calculations:

The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to the concentration of nitrogen that was in the original food (Equation 3). The following equation can be used to determine the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration:

 

Where vs and vb are the titration volumes of the sample and blank, and 14g is the molecular weight of nitrogen N. A blank sample is usually ran at the same time as the material being analyzed to take into account any residual nitrogen which may be in the reagents used to carry out the analysis. Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor: %Protein = F� %N.

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Description of the method: 

The method consists of heating a substance with sulfuric acid, which decomposes the organic substance by oxidation to liberate the reduced nitrogen as ammonium sulfate. In this step potassium sulfate is added to increase the boiling point of the medium (from 337°F to 373°F / 169°C to 189°C). Chemical decomposition of the sample is complete when the medium has become clear and colorless (initially very dark).

The solution is then distilled with sodium hydroxide (added in small quantities) which converts the ammonium salt to ammonia. The amount of ammonia present (hence the amount of nitrogen present in the sample) is determined by back titration. The end of the condenser is dipped into a solution of boric acid. The ammonia reacts with the acid and the remainder of the acid is then titrated with a sodium carbonate solution with a methyl orange pH indicator.

Apparatus:

 

Advantages and Disadvantages:

Advantages. The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. Its universality, high precision and good reproducibility have made it the major method for the estimation of protein in foods.

Disadvantages. It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts The technique is time consuming to carry-out.

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 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 the2008 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 (an hour or more), compare unfavorably with the Dumas method for measuring crude protein content.

Conversion factors

Conversion factors for common foods range from 6.38 for dairy and 6.25 for meat, eggs, corn (maize) and sorghum to 5.83 for most grains, 5.70 for wheat flour and 5.46 for peanuts.

Total Kjeldahl Nitrogen or TKN is the sum of organic nitrogen, ammonia (NH₃), and ammonium (NH₄⁺) in the chemical analysis of soil, water, or wastewater (e.g. sewage treatment plant effluent). To calculate Total Nitrogen (TN), the concentrations of nitrate-N and nitrite-N are determined and added to TKN.

TKN is determined in the same manner as organic nitrogen, except that the ammonia is not driven off before the digestion step.

The original TKN method was developed by the Danish chemist Johan Kjeldahl in 1883. Today, TKN is a required parameter for regulatory reporting at many plants but is also used to provide a means of monitoring plant operations.

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