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An Introduction to Kjeldahl Method

This method was specifically developed by Danish chemist, Johan G.C.T. Kjeldahl, in 1883 to determine the nitrogen contents in organic and inorganic substances  (like foodstuffs, fertilizers, wastewater, soil, feed, grain, and other substances). This method is also used for estimating the protein content in food.


Kjeldahl method is an analytical chemistry method that helps in the quantitative measurement and determination of nitrogen present in organic substances as well as inorganic compounds. The method was first developed by Johan Kjeldahl in the year 1883. This process plays an important role in the method of analyzing proteins. This method was developed to determine the nitrogen contents in organic and inorganic substances  (like foodstuffs, fertilizers, wastewater, soil, feed, grain, and other substances). This method is also used for estimating the protein content in food.

Applications of Kjeldahl Method

This method is used internationally as it is subjected to many applications which include precision and reproducibility. It is not however able to measure the true protein content. But still, it has a number of advantages in the field of chemical analysis.

The major applications of Kjeldahl nitrogen are as follows:

  1. Total kjeldahl nitrogen:

The total nitrogen found in organic substances is known as total Kjeldahl nitrogen. It is mostly used in sewage treatment plants effluent as it is helpful in the chemical analysis of soil water or wastewater. This method is the most accurate, versatile and efficient strategy.

Till now this particular treatment is used in many areas including treatment plants and as a means of monitoring plant operations.

  1. Conversion factors:

The total Kjeldahl nitrogen depends on the type of protein also. To work with total Kjeldahl nitrogen, it's important to note what fraction of the protein is composed of nitrogenous amino acids.

The range of conversion is very low. For food, it generally ranges from 6.38 for meat, egg, maize; it is 6.25 for sorghum; it is 5.83 for rice, and so on.

  1. Sensitivity :

The Kjeldahl method is not at all sensible in its original version. It needs to be made sensible by different processes which include potentiometric titration, zone capillary electrophoresis, and ion chromatography.

The Procedure involved in the Kjeldahl Method

The working principle of Kjeldahl analysis is three steps process as described below:

  1. Digestion: 

The organic sample provided or taken is firstly treated with a concentrated acid solution, mostly H2SO4. The solution is boiled at an extremely high temperature. The acid solution digests the sample to produce ammonium sulfate solution.

  1. Distillation:

The particular process is a combination of boiling and condensation. An excess of base is added to the formed solution to convert the ammonium sulfate solution to NH3 gas.

  1. Titration:

To finally quantify the nitrogen present in the sample, the obtained product from the previous process is titrated in order to give the final required results.

This particular method left a huge impact. Furthermore, studies have improved the quality of the method. Various scientific associations approved this method to be one of the most versatile methods. These associations include AOAC(Association Official Analytical Chemists), AACC(Association of American Cereal Chemists), AOCS(American Oil Chemist Society), EPA(Environmental Protection Agency), ISO(International Standards Organization). The following are the names of some of the associations which lead the scientific equipment for conducting the Kjeldahl method which is known as VELP.

Kjeldahl Method Reaction

  1. Digestion:

Organic (C, H, N) + H2SO4 → digest Cu2+ + (NH4)2SO4

  1. Distillation: 

(NH4)2SO4 + 2NaOH → Na2SO4 + 2H2O + 2NH3

And, NH3 + HCl → NH₄Cl

  1. Titration:

B(OH)2 + H2O + Na2CO3 → NaHCO3 + CO2 + H2

Note: Same steps are involved for protein estimation by the Kjeldahl method.

Kjeldahl Method Formula

The equivalent weight of NH3 is 17g/eq. And, 14 gm of Nitrogen is contained one equivalent weight of NH3. So, the percentage of nitrogen can be determined using the following formula:

Kjeldahl method of nitrogen estimation = 1.4V × NW


W = Weight of the sample used (in grams)

V = Acid used in titration (in ml)

N = Normality of standard acid

Limitations of Kjeldahl Method

This method measures only nitrogen bound to organic components (proteins, amino acids, nucleic acids) and ammonium in the sample. This method is not suitable for compounds containing nitrogen in azo and nitro groups or in rings (quinoline, pyridine, nitrate, and nitrite, etc). In these compounds, the nitrogen cannot be converted to ammonium sulfate following the Kjeldahl method.

Did You Know?

Protein quantification (total protein content in a sample) is necessary to understand the total protein content in a formulated product. Accurate protein quantification is important because a range of other evaluations require precise total protein content results in order to generate accurate data.

FAQs on Kjeldahl Method

1. What Does the Kjeldahl Method Measure?

Kjeldahl method is used for the quantitative determination of nitrogen contained in organic substances and the nitrogen contained in inorganic compounds like ammonium, ammonia.

2. What is the Kjeldahl Flask?

It is a round bottom flask with long wide necks that are used in the Kjeldahl method for the quantitative determination of a sample of nitrogen content. They are manufactured from borosilicate glass, which is resistant to heat and chemicals.

3. During the Estimation of Nitrogen Present in a Compound by Kjeldahl Method Calculation, the Ammonia Evolved from 0.5 g of the Compound in Kjeldahl's Estimation of Nitrogen Neutralized 10 mL of 1M H2SO4. Find Out the Percentage of Nitrogen Present in the Compound.

10 ml, 1M H2SO4 corresponds to 20 ml, 1M NH3.

⇒1000 ml of 1 M ammonia contains 14 g of nitrogen.

Hence, 20 ml of 1 M ammonia will contain (14 х 20)/1000 g of Nitrogen

⇒0.5 g of organic compound contains 14 х 20)/1000 g of Nitrogen.

Hence, 100 g of the compound will contain

(14 х 100 х 20) / (0.5 х 1000) = 56% Nitrogen.

4. What are the limitations of the Kjeldahl reaction?

Kjeldahl method is not applicable for compounds that contain nitro groups, azo groups or ringed groups as these are inconvertible to sulfates which hampers the further processes. These compounds are more stable and are considered less reactive.

5. What is the brief description of the method of the Kjeldahl reaction?

Kjeldahl reaction is mostly used to obtain nitrogen. The method involves the conversion of nitrogen into ammonium sulfate by digestion with sulfuric acid; later it alkalizes the solution and finally determines the ammonia obtained by the distillation process. As a final stage volume of standard acid is measured by titration.

6. How was the Kjeldahl reaction found?

In the year 1883, Johan Kjeldahl, experimented to produce nitrogen out of organic compounds. He started heating concentrated H2SO4(sulphuric acid) with organic samples, in a very high temperature that is 360 degree Celsius to 410 degree Celsius. This in return destroyed the organic sample and oxidized it to liberate the reduced nitrogen as ammonium sulfate. Later it is discovered that this particular experiment also works for inorganic samples.

7. What is the purpose of adding H2SO4 at the initial step of the Kjeldahl method?

This method is fully based on a high boiling point. Salt and catalyst highly increase the speed of digestion. In addition, if potassium sulfate is added that increases the boiling point of sulfuric acid furthermore. Finally, the action of the catalyst makes the speed and efficiency of the digestion procedure more rapid, henceforth improving the analytical method.

8. What is the function of mercury in the Kjeldahl method?

With the passage of time many changes have been recorded in order to get a more preferable method a new technique termed by the AOAC (Association Official Analytical Chemists)  has been introduced which favours micro Kjeldahl as well as macro Kjeldahl both. This process uses Mercury as a catalyst in order to trap ammonia then the process continues as such and later the standard acid solution is titrated against sodium hydroxide.

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