Kjeldahl Method: Need, Procedure, Findings

The Kjeldahl method has a wide application in biochemistry. We can test the Kjeldahl method in any substance that we came up with in our life to check the presence of organic and inorganic nitrogen, and eventually the presence of protein. Many research centers, such as ADAC and US EPA have taken the Kjeldahl method as an approved method for recognizing the nitrogen presence and its quantity in many organic and inorganic substances. But how is this Kjeldahl method performed? What are the different steps and apparatus required in the Kjeldahl method? Have a detailed look at these aspects in this article.

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Kjeldahl method and its need

The Kjeldahl method, named after its inventor Johan Kjeldahl, is a method that is more than a hundred years old. Even today, this method has not been replaced in many chemistry labs. Mainly Kjeldahl method is performed for two things:

• Determination and measurement of nitrogen and organic substances like soil, meat, fish, etc.
• Determination of protein presence.

When compared with many other methods, the Kjeldahl method excels in the measurement of nitrogen content in soil samples. This makes the Kjeldahl method highly applicable in soil research centers and agricultural institutes to analyze the fertility of soils. Also, this method is the most relied method for determining the protein content of food samples.

We know that nitrogen is often present in the form of inorganic compounds in many substances. In such cases, the Kjeldahl method fails as it cannot be converted into ammonium sulfate. This is one shortcoming of the Kjeldahl method. For the same reason, it is no use for the determination of nitro, azro groups, and nitrogen present in the form of rings. Many cases in which the Kjeldahl method fails to analyze protein are also seen. But generally, the Kjeldahl method is regarded as the standard method of nitrogen determination, irrespective of these shortcomings.

The video below explains this:

Kjeldahl Method Detailed Video Explanation:

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Procedures of Kjeldahl method

The Kjeldahl method is performed in three steps, namely digestion, distillation, and titration.  We can try the Kjeldahl method with a sample for the test (nitrogen content), a Kjeldahl flask (a heat resistant, long-necked flask made of borosilicate glass, used in experiment labs) a condenser, and some basic laboratory equipments.

These are the three steps involved:

Digestion

The primary stage of the Kjeldahl method is digestion. Digestion simply means the conversion of organic nitrogen into NH4+ (Ammonium ions). This is done by breaking down the nitrogen bonds into Ammonium ions. In the process, the sample is transformed into the black foam with the presence of carbon. The sample is heated in the presence of catalysts like Potassium Sulphate, and heated at a high temperature to fasten the digestion process.

This can be denoted as:

Protein (-N) + H2SO4→ (NH4)2SO4+CO2+H2O

Distillation

The digested sample is allowed to cool down at room temperature, before taking it for the next process, i.e., distillation process.  In this process, the NH4+ we obtain during digestion is converted into Ammonia. NaOH (alkali) acts as an agent for this purpose. From the heated NH4+ ions, a tube connects to the receiving vessel which is full of solutions that can absorb ammonia gas (usually, B(OH)3 or HCL).

This can be denoted as (when aqueous boric acid is used)

B(OH)3 + NH3+ H2O↔NH4+ + (OH)4-

Titration

Finally, the concentration that is obtained as the result of distillation is used to find the nitrogen presence and measure it. This process is called titration. Titration is of two types:

• Direct titration

Here, we use a mixture of indicators with acids like sulphuric acid and HCL for titration. Concerning the amount of ammonium ions present in the concentration, the mixture is used at a range of 0.01N-0.5N.

This can be denoted as (When HCL is used)

B(OH)4- + HCL ↔Cl- + B(OH)3+ H2O

• Back titration

In a back titration, the sulphuric acid residues obtained by using sulphuric acid as the absorbing solution are titrated with sodium hydroxide which helps in calculating the difference in the ammonia presence.

This can be denoted as:

H2SO4+ 2NH3→SO4 2- + 2NH4+

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Reagents Used in the Kjeldahl Method

Reagents have an important role in the process of the Kjeldahl method. The selection of reagents depends on the type of sample that is used to determine the presence of nitrogen. The most commonly used reagents of the Kjeldahl method includes:

Catalyst

During the digestion process, catalysts are used to increase the speed and efficiency of the process. The selection catalysts depend on the substance that is used for the Kjeldahl method. Copper is the most common catalyst used in the Kjeldahl method.

Acids and Oxidants

Acids and oxidant agents are used to increase the speed of the digestion process. It accelerates the decomposition of organic material and reduces foam during digestion.

Alkali

Alkali (NaOH) helps in neutralizing the acidity of the sample, before the distillation of the sample in the Kjeldahl method. It boosts the ammonium recovery during the distillation process.

Ammonia capturing solutions

During distillation, the presence of an ammonia receiving solution is inevitable to capture the ammonia gas. Acids like Boric acid, sulphuric acid, and HCL are used for this purpose.

Indicators

Indirect titration, if boric acid is used as the receiving solution, tetrahydroxyborate anions are formed .This is detected with the help of combining indicators like methyl red and methylene blue. In the case of back titration, color indicators are used for detection.

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Findings of the Kjeldahl method

After performing the above steps of the Kjeldahl method, the following formula is used to determine the percentage of nitrogen in a sample:

1.4x volume of acid in titration x normalityWeight of sample          

(N.B: here, normality refers to the normality of the standard acid used. The volume of acid would be calculated in milli-liter and the weight of the sample in grams)

Studies done using the Kjeldahl method on different substances have secured different results. Brown rice shows 1.3 % of nitrogen, where wheat grains show 2.4 % nitrogen and they both have 7.9 % and 13.7 % of proteins respectively. You can find a few more examples in the slideshow below.

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Things to Remember

• The Kjeldahl method is an approved method for recognizing the nitrogen presence and its quantity in many organic and inorganic substances.
• The Kjeldahl method helps in the determination and measurement of nitrogen and organic substances like soil, meat, fish, etc... And also helps in the determination of protein presence.
• The Kjeldahl method is performed by three steps, namely- digestion, distillation, and titration.
• Digestion is the process in which the conversion of organic nitrogen into NH4+ (Ammonium ions) happens. This is done by breaking down the nitrogen bonds into Ammonium ions. In the process, the sample is transformed into a black foam with the presence of carbon.
• Through the distillation process, the NH4+ we obtained during digestion is converted into Ammonia. NaOH (alkali) acts as an agent for this purpose. From the heated NH4+ ions, a tube connects to the receiving vessel which is full of solutions that can absorb ammonia gas.
• After distillation, the concentration that is obtained as the result of distillation is used to find the nitrogen presence and measure it through the titration process.

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