Summary notes - The pauly test and procedure PDF

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Summary notes - The pauly test and procedure The Pauly’s test is a biochemical test for the detection of tyrosine and histidine where the reagent couples with amines, phenols, and imidazole groups. The test was discovered by and is named after the German Chemist Hermann Pauly. The Pauly’s test is a specific test for proteins containing tyrosine and histidine as the reagent undergoes diazotization with aromatic groups. The reaction is performed under cold conditions as the diazonium compound can only form at cold temperatures. The test is a specific test for proteins, which is performed for further differentiation between amino acids and proteins.

The Objectives of Pauly’s Test To detect the presence of tyrosine and histidine-containing proteins. To differentiate between histidine and tyrosine from other amino acids.

The Principle of Pauly’s Test Pauly’s test is based on the principle of coupling between the amino acids and the diazonium ion formed in the reagent. The pauly’s reagent consists of sulphanilic acid dissolved in concentrated hydrochloric acid. The sulfanilic acid undergoes diazotization in the presence of sodium nitrate and hydrochloric acid. The result of the diazotization reaction is the diazonium salt (p-phenyldiazosulphonate). The diazonium salt thus formed couples with histidine molecule in an alkaline condition to form a dark red or cherry colored compound. 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. This is 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.

Reaction Sulphanilic acid + HNO2 + HCl

Diazonium salt + Tyrosine

Requirements

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Diazonium salt + 2H2O

Azo dye (red color)

Reagent

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

Ice bath Vortex Test tubes Test tube stand Pipettes

The Procedure of Pauly’s Test To a test tube, 1 ml of chilled sulfanilic acid is added, and a few drops of pre-chilled sodium nitrite are mixed in a vortex. One ml of amino acid sample is immediately added to the test tube and mixed in a vortex. Few drops of sodium carbonate are added to the test tube drop by drop until color begins to appear....