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 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 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
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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.

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

measured measured
ammonia ammonium
excess sodium
sulfate sulfate
acid hydroxide
(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 boric ammonium- excess

gas acid borate complex 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-
sulfuric ammonium boric
borate
acid sulfate acid
complex
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.

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