The document describes the Kjeldahl method for determining nitrogen content in organic and inorganic substances. It was developed in 1883 and involves three main steps: digestion using sulfuric acid to convert organic nitrogen to ammonium ions, distillation to separate ammonia from the digestion mixture, and titration to quantify the ammonia content. The Kjeldahl method is widely used to analyze samples like wastewater, soils, foods and fuels to determine their nitrogen, protein or nutrient content. While it remains the standard, it is not suitable for all types of nitrogen compounds and accuracy can be affected by factors like digestion time and non-protein nitrogen content.

1. Industrial Pollution Control Lab By: Kawar Abid
Determination of Nitrogen in the Wastewater By Kjeldahl Method
Theory
Nitrogen is one of the five major elements found in organic materials such as protein. This fact was
recognized by a Danish chemist, Johan Kjeldahl, This method was specifically developed for
determining the nitrogen contents in organic and inorganic substances. In 1883 Kjeldahl presented
to the Danish Chemical Society a method (much revised since his day) for determining the amount
of nitrogen in mixtures of substances containing ammonium salts, nitrate, or organic nitrogen
compounds.
The central basis used in this procedure is the oxidation of the organic compound using strong
sulfuric acid. As the organic material is oxidized the carbon it contains is converted to carbon
dioxide and the hydrogen is converted into water.
The nitrogen, from the amine groups found in the peptide bonds of the polypeptide chains, is
converted to ammonium ion, which dissolves in the oxidizing solution, and can later be converted
to ammonia gas.
In today’s world, Kjeldahl nitrogen determinations are used on several samples like wastewater,
industrial water, fossil fuels, soil, fertilizers, meat, feed, grain, and many other substances. The
Kjeldahl method of nitrogen analysis is the worldwide standard for calculating the protein content
in a wide variety of materials ranging from human and animal food, fertilizer, waste water and
fossil fuels.
The Process of Kjeldahl Method
Since the development of Kjeldahl method about 100 years ago the technique and apparatus have
been modified and made better. However, even with the new changes the basic principles
introduced by Johan Kjeldahl is still relevant. The main objective or purpose of this procedure is
the oxidation of organic compounds using concentrated sulphuric acid.

2. Industrial Pollution Control Lab By: Kawar Abid
Overall, the Kjeldahl method is divided into three main steps. The method has to be carried out in
proper sequence. The steps include digestion, distillation, and titration.
1. Digestion:
This is the most time-consuming step in the analysis. The purpose of this step is to break down
the bonds that hold the polypeptides together, and convert them to simpler chemicals such as
water, carbon dioxide and, of course, ammonia.
Such reactions can be considerably speeded up by the presence of a catalyst and by a neutral
substance, such as potassium sulfate (K2SO4), which raises the boiling point of the digesting
acid and thus the temperature of the reaction.
Catalysts are also used to help in the digestion process; many different one have been tried
including selenium, mercury, copper, or ions of mercury or copper.
Organic compound+H2SO4→[digest]Cu2
+(NH4)2SO4
2. Distillation: The purpose of the next step, distillation, is to separate the ammonia (that is, the
nitrogen) from the digestion mixture. This is done by,
1. By raising the pH of the mixture using sodium hydroxide (40% NaOH solution). This has the
effect of changing the ammonium (NH4
+
) ions (which are dissolved in the liquid) to
ammonia (NH3), which is a gas.
(NH4)2SO4+2NaOH Δ
→ Na2SO4+2H2O+2NH3
2. Separating the nitrogen away from the digestion mixture by distilling the ammonia
(converting it to a volatile gas, by raising the temperature to boiling point) and then trapping
the distilled vapors in a special trapping solution of Boric Acid ( NH3 gas again convert to
NH4
+
ion)
H3BO3+ NH3 → NH4 + + H2BO3
-
The majority of the NH3 is distilled and trapped in the receiving acid solution within the
first 5 or 10 minutes of boiling. But depending on the volume of the digestion mixture
and the method being followed, 15 to 150 ml of condensate should be collected in the
receiving flask to ensure complete recovery of nitrogen.
3. Titration: There are two types of titration: back titration, and direct titration. Both
methods indicate the ammonia present in the distillate with a color change and allow for
calculation of unknown concentrations.

3. Industrial Pollution Control Lab By: Kawar Abid
The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the ammonia
collects, the color of the receiving solutions changes.
NH4 + H2BO3 + HCl → NH4Cl + H3BO3
Limitations of Kjeldahl Method
While the Kjeldahl method of nitrogen analysis has become the worldwide standard, this method is
not suitable for compounds containing nitrogen in azo and nitro groups or in rings (quinoline,
pyridine, etc.). In these cases, the nitrogen cannot be converted to ammonium sulphate by following
the Kjeldahl method.
Kjeldahl Method PROCEDURE
1) Digestion
1- Sample: 20 mL of water sample in a Kjeldahl flask.
2- Reagents for digestion add to the sample: Use Copper Sulfate as catalyst, Potassium Sulfate to
increase boiling point
With ratio 5:3 of Potassium Sulfate (K2SO4) + Copper Sulfate (CuSO4)
3- Add 1 g of prepared reagent above to Kjeldahl flask.
4- Add 20 mL of concentrated Sulfuric acid (H2SO4) to Kjeldahl flask.
5- heat the mixture for 60 – 120 minutes, till the sample getting clear (no smoke) and green color.
6- Cooling and diluting: let the digestion flask to cool in room temperature and dilute it with
distilled water to be 100 mL of final volume.
2) Distillation
1- Measure 30 mL of Boric Acid 4% into and pour this acid to conical flask. Place this conical flask
in the distillation unite where the distillate will be collected. (output of distillated will be immersed
directly into this flask which contain boric Acid)
2- Add 10 mL of diluted digestive juice (point 6 from Digestion) to the distillation.
3- Add 50 mL of Sodium Hydroxide 40% to the distillation. pH will raise to be (PH>10)

4. Industrial Pollution Control Lab By: Kawar Abid
4- Finally add 50 mL of D.W to the distillation.
5- Run the distillation and heat it to 150-200 Co
.
6- Collect approximately 100 mL of distillate. (Boric Acid with distillate reach to be 100 mL).
3) Titration
- Take 0.1 N of standardized Hydrochloric Acid HCl in burette.
- Add a few drops of Methyl Red to conical flask with mixture (form point 6 Distillation which
contain 100 mL of distilled ammonia with boric acid). The color will change from transparent to
yellow.
- Titrate the distillate with 0.1 N of HCl till the color change from yellow to orange.
CALCULATIONS:
One mole of ammonia coming from the digestion mixture (and hence from the original sample)
will neutralize exactly one mole of the acid in the trapping flask.
The first calculation, therefore, is to find the number of moles of ammonia that have been produced
and then trapped from your sample(s).
This is done by, moles of ammonia = moles of acid (used in the burette for titration)
N% =
mL of Titrant ∗ Noramlity of Titrant (HCl)∗ Acid Factor ∗ M.w of N
(V1 or W1) ∗ F
N: Nitrogen percentage
Acid Factor: The number of proton H+
donated during ionization
HCl ionized with NaOH and gives 1 proton H+
here n-factor will be 1
V1: mL of polluted sample used
W1: grams of sample used
If your sample is solid will use W1 as grams while if it liquid will use V1 as a mL.

5. Industrial Pollution Control Lab By: Kawar Abid
F: Dilution Factor =
Total mL of Digested Juice
mL used for Distillation
We used 10 ml for the distillation from total100 ml of digested juice
F =
100 mL
10 mL
= 10
M.w of N: 14.0067
For farther knowledge the N% ratio can be used to determine the amount of crude protein
in sample as follow:
Crude Protein%: N% * F * Factor
F: Dilution Factor, it was 10
Factor: each kind of known product sample has its known factor
For example:
For Milk product is 6.35
Wheat grains is 5.70
Forages and Feed is 6.25
Questions:
1- Are there any alternative methods for nitrogen determination, and how do they
compare to the Kjeldahl method?
2- What are some common challenges or sources of error in the Kjeldahl method?
3- Why is sulfuric acid used in the digestion step of the Kjeldahl method?
4- Explain the rule of each chemical used in this method.
5- What type of samples is the Kjeldahl method commonly used for?
6- How does the choice of a different indicator for titration in the Kjeldahl method (e.g.,
methyl orange vs. bromothymol blue) affect the precision and sensitivity of the
analysis?
7- What are the factors influencing the release of non-protein nitrogen compounds during
the Kjeldahl digestion, and how can these be minimized to improve method accuracy
for protein determination?
8- How does the duration of the digestion step in the Kjeldahl method impact the
accuracy and precision of nitrogen determination in samples?

6. Industrial Pollution Control Lab By: Kawar Abid