Tollen’s test or Silver mirror test: Principle, Requirements, Procedure, Result Interpretation and Limitations

June 8, 2021 Sushil Humagain Biochemistry, Chemistry, Laboratory tests, Microbiology 0

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  • Tollen’s test is a chemical test used to distinguish reducing sugars from non-reducing sugars.
  • It is also known as silver mirror test since free silver metal is formed at the end of this test reaction.
  • It also helps in the differentiation of aldehydes and ketones through routine qualitative organic analysis.

Principle:

  • Reducing sugars or aldehydes react with Tollen’s reagent (ammoniacal solution of silver nitrate) to reduce it to form free silver metal.
  • Tollen’s reagent is an alkaline solution of silver nitrate (AgNO3) mixed with liquid ammonia (NH3) which results in the formation of a complex.
  • Brown ppt of silver oxide (Ag2O) is formed when AgNO3 reacts with NaOH.
  • Silver oxide is then dissolved by aqueous ammonia to form a complex, [Ag(NH3)2]NO3. 
  • This complex in Tollen’s reagent is a strong oxidizing agent that oxidizes the aldehyde group present in some carbohydrates to form a carboxylic acid.
  • The silver ions present in the reagent are reduced to free metallic silver which forms a silver mirror on the bottom and sides of the test tube.
  • Ketones generally give negative Tollen’s test. However, an α-hydroxy ketone gives a positive test since it is oxidized into an aldehyde by Tollen’s reagent. 

Principle reactions:

2AgNO3 + 2NaOH    →    Ag2O (brown ppt) + 2NaNO3 + H2O

Ag2O (brown ppt) + 4NH3 + 2NaNO3 + H2O    →    2[Ag(NH3)2]NO3 + 2NaOH

Glucose + 2[Ag(NH3)2]NO3 + H2O    →    2 Ag(silver mirror) + 4 NH3 + Gluconic acid + 2H+

Requirements:

  • Sample solution
  • Tollen’s reagent (Ammoniacal solution of silver nitrates)
  • Test tubes
  • Pipette
  • Water bath

Procedure:

  • First of all, two clean and dry test tubes are taken and labeled as test (T) and blank (B).
  • 1 ml each of sample solution and distilled water are pipetted into tubes T and B respectively.
  • 2 ml of Tollen’s reagent is added to both the test tubes.
  • The test tubes are kept in warm water bath for 3-5 minutes.
  • The color formation is observed and noted down.

Result interpretation:

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  • If a dark grey precipitate or silver mirror is formed on the bottom and sides of the test tube, it indicates a positive result. It confirms the presence of reducing sugars/ aldoses/ α-hydroxy ketoses in the sample solution.
  • The absence of such precipitate means a negative result. i.e. absence of reducing sugars/ aldoses/ α-hydroxy ketoses in the sample solution.

Limitations:

  • Tollen’s reagent must be prepared immediately prior to its use as an explosive substrate can be formed if it is allowed to dry.
  • The glassware must be rigorously cleaned with concentrated KOH (Potassium Hydroxide) because if they are not perfectly clean, a dark precipitate instead of grey may appear in the solution.
  • Some carbohydrates devoid of aldehyde group might give a positive result because of the isomerization of such sugars under alkaline conditions.

Tollen’s test or Silver mirror test: Principle, Requirements, Procedure, Result Interpretation and Limitations