The Kjeldahl method was developed by a brewer called Johann Kjeldahl in 1883. The protocol is built on the principle that strong acid helps in the digestion of food so that it releases nitrogen which can be determined by a suitable titration technique. By observing the nitrogen concentration of the food, the amount of protein present is then calculated. This method is particularly ideal for insoluble proteins, protein in foods and protein covalently immobilized on chromatographic supports.


  • selenium reaction mixture for nitrogen determination according to Wieninger
  • sulfuric acid (98% w/w)
    • Sulfuric Acid (Used to digest the samples – )
  • 60% NaOH, 10% Na2S2O3 (w/v) in ddH2O
    • Sodium Hydroxide Reagent (For the dilution of reagents – )
    • Distilled Water (Used in the dilution of reagents – )
  • 2% boric acid (w/v) in ddH2O
    • Boric Acid (Used in the preparation of solutions – )
  • Tashiro indicator (2 vol. 0.2% methyl red in 90% ethanol + 1 vol. 0.2% methylene blue in 90% ethanol)
  • 010 N HCl (standard solution)
    • Hydrochloric Acid (It activates pepsinogen and converts it to the enzyme pepsin – )

Preparation of Reagents and Experiment Protocol:

Step 1:

Take 1.5 g catalyst A and mix it with accurately weighed 100–250 mg sample. After that, add 3 ml of concentrated sulfuric acid B.

Step 2:

At the temperature of boiling sulfuric acid (about 180 ◦C), heat the mixture for 2 hours. Note that the acid condenses in the middle of the neck of the Kjeldahl flask.

Step 3:

After cooling put the flask into the distillation apparatus; after that slowly add 12 ml ddH2O followed by 12 ml of Solution. C.

Step 4:

To nearly 100◦C heat the mixture to liberate ammonia which is distilled by steam for about 10 minutes through a condenser, the tip of which is submerged in a flask containing 5 ml of Solution D.

Step 5:

After the distillation is finished (total volume about 25 ml), titrate the ammonia with Soln. F. followed by the addition of three drops of E.


The results are calculated as follows:

1.0 ml 0.010 N HCl = 10 µMol N = 0.14 mg N

By means of the Kjeldahl factors F the amount of protein is:

mg protein = mg N · F and the protein content “c” of the sample;

c [% ] = (mg protein) · 100
weight of sample Since no additional incubations or reagents are required, quantifying protein by directly measuring absorbance is fast and convenient. No protein standard needs to be prepared, and the procedure does not consume the protein. The relationship of protein concentration to absorbance is linear. When different dilutions of a compound are compared, this should be taken into consideration. By reading the UV absorption, the concentration of an aqueous protein solution can be estimated.

With the absorbance maxima at 280 and 200 nm proteins in solution absorb the ultraviolet light. For the absorbance peak at 280 nm, amino acids with aromatic rings are the primary reasons for that. Peptide bonds are usually responsible for the peak at 200nm -215 nm. Protein structures like secondary, tertiary, and quaternary all affect absorbance; therefore factors such as ionic strength, pH, etc. can change the absorbance spectrum.

The measurements of the absorbances have to be done against the protein-free solvent (buffer). The absorbance of this blank has to be subtracted from that of the protein solution if a single-beam photometer is used. The following equations are made for 10.0 mm:

  • Warburg and Christian equation:

mg protein/ml = 1.55 · A 280− 0.76 · A 260

This estimation of protein concentration is valid up to 20% (w/v) nucleic acid or an A280/A260 ratio < 0.6.

  • Kalckar and Shafran equation:

mg protein/ml = 1.45 · A 280 − 0.74 · A 260

  • Whitaker and Granum equation:

mg Protein/ml = (A235− A 280) : 2.51

  • The concentration of immunoglobulins:

mg IgG/ml = A280 : 1.38

  • Beer–Lambert law:

Aλ = log10 I0 = ελ · c · d


Aλ: absorbance at wavelength λ;

I0: the intensity of incident light;

I: the intensity of transmitted light;

ελ: (molar) absorption coefficient at wavelength λ;

c: concentration;

d: length of optical path within the cuvette.

Similarly, if solutions of pure proteins with known amino acid sequence or composition are measured, the concentration c (mol/l) is calculated from the absorbances at 280 nm (A280), 320 nm (A320), 350 nm (A350), and the number of tryptophan (nTrp) and tyrosine residues (nTyr) and the number of disulfide bridges (nS−S) according to the following equation:

c =   ___E280 − 10(2.5 · lgE320−1.5 · E350)
5540 · nTrp + 1480 · nTyr + 134 · nS−

  1. Stoscheck, CM. (1990). Quantitation of protein. Methods Enzymol. 182:50-68.
  2. Whitaker, JR., Granum, PE. (1980). An absolute method for protein determination based on difference in absorbance at 235 and 280 nm. Anal Biochem; 109(1):156-9.
  3. Harris, DA. CL, Bashford. (1987). Spectrophotometric assays. In, eds, Spectrophotometry and Spectrofluorimetry: a Practical Approach. IRL Press, Oxford, pp 59-61
  4. K., Smith., R.I., Krohn., G.T., Hermanson et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem;150(1):76-85.