Pauly’s Test: Principle, Reaction, Reagent, Procedure And Results

What Is Pauly’s Test?

Pauly’s test is a chemical test to detect the amino acid histidine and tyrosine, this test is named after the German chemist Hermann Pauly, who first described it. In this test, when proteins containing either tyrosine or histidine are reacted with diazotized sulfanilic acid under alkaline conditions to form dark red compound. The reaction is conducted under cold conditions since diazonium compound can only form at cold temperatures.


  • To detect the presence of tyrosine and histidine-containing proteins.
  • To differentiate between histidine and tyrosine from other amino acids.

Pauly’s Test Principle

 This test is specific for the detection of Tryptophan or Histidine. Pauly’s test is premised on the principle of coupling reaction between the amino acids and the diazonium ion present in the reagent. The reagent used for this test contains sulphanilic acid dissolved in hydrochloric acid. Sulphanilic acid upon diazotization in the presence of sodium nitrite and hydrochloric acid results in the formation a diazonium salt (p-phenyldiazosulphonate).  The diazonium salt formed couples with either tyrosine or histidine in alkaline medium to give a dark red or cherry colored chromogen (azo dye). The color might decrease in intensity and changes into an orange color when the solution is made acidic. In the case of tyrosine, a similar cherry red-colored complex is formed which then changes into yellow color on dilution and bronze-yellow color on acidification. Each molecule of histidine and tyrosine reacts with two moles of diazonium compound reacts to form a mole of bis(azobenzenesulphonic acid)histidine or bis(azobenzenesulphonic acid)tyrosine.

Summary reaction:

  • Sulfanilic acid + sodium nitrite + sodium carbonate = diazonium component
  • Diazonium component + (imidazole ring of histidine / phenol group of tyrosine) = dark red color solution


Reagent And Material Required


  • 1% Sulphanilic acid (chilled)
  • 10% HCl
  • 5% Sodium nitrite (chilled)
  • 10% Sodium carbonate
  • Chilled sample (1% tyrosine, 1% histidine)

Material Required

  • Ice bath
  • Vortex
  • Test tubes
  • Test tube stand
  • Pipettes

Pauly’s Test Procedure

  1. Put 2 ml of the test solution or sample to be tested in a test tube, cooled in ice box.
  2. Add 1ml of sulphanilic acid, mix well and keep in ice bath.
  3. Now add 1ml sodium nitrite solution to all test tubes.
  4. Leave in ice bath for 3 minutes.
  5. Make the solution alkaline by adding 5ml of sodium carbonate.
  6. Observe the color changes.

Pauly’s Test Result Interpretation

  1. Positive Test: A positive result is indicated by the appearance of a red-colored complex, which is a confirmation of the presence of histidine and tyrosine in the solution.
  2. Negative Test: A negative result is indicated by the absence of a red-colored solution, which is a confirmation of the absence of histidine and tyrosine in the sample.

Limitations of Pauly’s Test

  • The formation of diazonium salt occurs at cold temperatures; thus, the test has be performed in the presence of ice for accurate results.
  • The test doesn’t allow the differentiation between histidine and tyrosine. An extra test such as millon’s test is required for the differentiation. Millon’s Test can be performed as histidine gives a negative result in Millon’s test.