Kjeldahl Method

• Method for quantitative determination of Nitrogen

• Developed by Johan Kjeldahl in 1883.
• Universal, Précised and Reproducible method
for estimating the protein content in foods.
• Also used to assay nitrogen content in soils, waste waters,
fertilizers and other materials.
• Does not give a measure of true protein content, as it
measures non protein nitrogen in addition to the nitrogen in
proteins
 Kjeldahl method - three steps
• the sample is first digested in strong sulfuric acid in the
presence of a catalyst, which helps in the conversion of
the amine nitrogen to ammonium ions,
• the ammonium ions are then converted into ammonia
gas, heated and distilled. The ammonia gas is led into a
trapping solution where it dissolves and becomes an
ammonium ion once again,
• finally the amount of the ammonia that has been
trapped is determined by titration with a standard
solution, and a calculation made.
 Kjeldahl Method: Principle
1. Digestion in presence of strong sulfuric acid
2. Distillation (to liberate of Ammonia gas)
3. Back-titration

http://people.umass.edu/~mcclemen/581Proteins.html
 Digestion
• Weighing out approx 1 gm of the sample containing
protein, making a note of the weight, and placing the
sample into a digestion flask, along with 12-15 ml of
concentrated sulfuric acid (H2SO4).
• Adding seven grams of potassium sulfate and a catalyst,
usually copper. ( speed up the reaction by increasing BP)
• Bringing the digestion tube/flask and mixture to a "rolling
boil" (about 370oC to 400oC) using a heating a block.
• Heating the mixture in the tube/flask until white fumes can
be seen, and then continuing the heating for about 60-90
mins.
• Cooling the tube/flask and cautiously adding 250 mL of
water.
 Distillation
• Tthe pH of the mixture is raised using sodium
hydroxide (45% NaOH solution). This has the effect of
changing the ammonium (NH4+) ions (which are
dissolved in the liquid) to ammonia (NH3) gas.
• the ammonia is distilled from the digestion mixture
(converting it to a volatile gas, by raising the
temperature to boiling point) and then trapping the
distilled vapors in a special trapping solution containing
boric acid
• removing the trapping flask and rinsing the condenser
with water so as to make sure that all the ammonia has
been dissolved.
 Back titration

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.
 Direct titration
boric acid is used as the receiving solution

The boric acid captures the ammonia gas, forming an ammonium-borate complex. As the
ammonia collects, the color of the receiving solutions changes.
• The nitrogen content is then estimated by titration of the ammonium borate
formed with standard sulfuric or hydrochloric acid, using a suitable indicator to
determine the end-point of the reaction.
• H2BO3- + H+ H3BO3 (4)
• 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
• 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 = 6.25 %N.
 Kjeldahl Method: Principle
1. Digestion:
Sample + H2SO4 → (NH4)2SO4(aq) + CO2(g) + SO2(g) +
H2O(g)
2. Liberation of ammonia:
(NH4)2SO4(aq) + 2NaOH → Na2SO4(aq) + 2H2O(l) +
2NH3(g)
3. Back-titration:
B(OH)3 + H2O + NH3 → NH4+ + B(OH)4–
B(OH)3 (excess) + H2O + Na2CO3 → NaHCO3(aq) +
NaB(OH)4(aq) + CO2(g) + H2O