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Pauly’s Diazo Test for Amino Acids

In this Pauly’s test for amino acids post we briefly summarises about: principle, reagents requirements, Pauly’s test procedure, result, application and limitations of Pauly’s test.

Pauly’s Diazo Test for Amino Acids

Pauly’s test is a biochemical test for the detection of tyrosine and histidine where the reagent couples with amines, phenols, and imidazole groups. Hermann Pauly, a German chemist, discovered the test and named it after him. Because the reagent is diazotized with aromatic groups, Pauly’s test is selective for proteins containing tyrosine and histidine. 

Because the diazonium molecule can only form at low temperatures, the reaction is carried out in a cold environment. Pauly’s test is a protein-specific test that is used to further distinguish between amino acids and proteins.


To distinguish between histidine and tyrosine from other amino acids and identify the presence of tyrosine and histidine-containing proteins.


Pauly’s test works on the premise of amino acid coupling to the diazonium ion generated in the reagent. Sulphanilic acid is dissolved in strong hydrochloric acid to make Pauly’s reagent. 

In the presence of sodium nitrate and hydrochloric acid, sulfanilic acid is diazotized. The diazotization reaction produces the diazonium salt (p-phenyldiazosulphonate). 

In an alkaline environment, the diazonium salt forms a dark red or cherry-colored combination with the histidine molecule. When the solution is made acidic, the colour may fade and shift to an orange colour. 

Tyrosine forms a similar cherry red-colored complex, which changes to yellow hue with dilution and bronze-yellow colour with acidification. 

Each molecule of histidine and tyrosine combines with two moles of diazonium compound to generate bis (azobenzenesulphonic acid) histidine or bis (azobenzenesulphonic acid) tyrosine.



1. 1% Sulphanilic acid (chilled)

2. 10% HCl

3. 5% Sodium nitrite (chilled)

4. 10% Sodium carbonate

4. Chilled sample (1% tyrosine, 1% histidine)


1. Ice bath

2. Vortex

3. Test tubes

4. Test tube stand

5. Pipettes


1. To 0.5 ml of 0.5% sulfanilic acid add an equal volume of 0.5% freshly prepared sodium nitrite. Allow to stand for 1 minute and add 1 ml of protein solution. Mix well and add 1 ml of 10% Na2CO3 to make the solution alkaline.


Pauly’s Test

Pauly’s Diazo Test for Amino Acids

Cherry red or orange red color may be observed. Cherry red color indicates presence or predominance of histidine and orange red color shows the presence or predominance of tyrosine in the solution

Positive result: The presence of histidine and tyrosine in the solution is shown by the emergence of a red-colored complex, suggesting a positive result.

Negative result: The absence of a red-colored solution indicates the absence of histidine and tyrosine in the sample, indicating a negative result.


1. Pauly’s test is used to detect the presence of proteins containing tyrosine and histidine, as well as to distinguish histidine and tyrosine from other amino acids.


1. Because diazonium salt is generated at low temperatures, Pauly’s test should be conducted in the presence of ice.

2. Pauly’s test is unable to distinguish between histidine and tyrosine. Millon’s Test can be used since histidine results in a negative result.

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