Barfoed’s Test: Objective, Principle, Procedure, Results And Application

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What Is Barfoed’s Test?

Barfoed’s test is a biochemical test used to detect monosaccharide (reducing) sugars in solution. The technique was devised by a Swedish physician C. T. Barfoed (1815–1899). Barfoed’s reagent, a mixture of ethanoic (acetic) acid and copper(II) acetate, is added to the test solution and boiled. If any reducing sugars are present a red precipitate of copper(II) oxide is formed. The reaction will be negative in the presence of disaccharide sugars as they are weaker reducing agents. The test is similar to the reaction of Fehling’s solution to aldehydes.

Objective

  • Barfoed’s test is done to distinguish between monosaccharides and reducing disaccharides.

Principle Of Barfoed’s Test

Barfoed’s test reaction is based on the reduction of cupric acetate by reducing monosaccharides and reducing disaccharides. Reduction of cupric acetate produces cuprous oxide which gives a brick red precipitate. RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+

Monosaccharides usually react in about 1-5 min and produce a red precipitate. While the reducing disaccharides take a much longer time between 7-12 min to give a red precipitate. The reaction with disaccharides is slower because disaccharides have to get hydrolyzed first and then react with the reagent cupric acetate to produce cuprous oxide. The difference in the time of appearance of precipitate thus helps distinguish reducing monosaccharides from reducing disaccharides.

Experiment

Materials And Reagents Required

  • Copper acetate: Provides an acidic medium
  • Acetic acid: Provide cupric ions
  • Test solution: 5% Glucose, 5% Sucrose, 5% Maltose, 5% Lactose, 5% Starch
  • Water bath
  • Barfoed’s reagent: Dissolve 13.3g of copper acetate in 200 ml of distilled water and add 1.8 ml of glacial acetic acid to it.
  • Dry test tubes
  • Pipettes

Barfoed’s Test Procedure

  1. Take 1 ml of a given sample in a clean, dry test tube. The concentration of disaccharides sample (if used) should not exceed 1% (w/v).
  2. Take control of 1 ml of distilled water in another tube.
  3. Add about 2-3 drops of Barfoed’s reagent to both the tubes and mix them in a vortex.
  4. Keep the test tubes in the water bath for 1-2 minutes. The boiling should not be done for more than 2 minutes as the disaccharides might hydrolyze into monosaccharides and give a positive result.
  5. Observe the appearance of color in the test tubes.
  6. Noted own the time taken for the appearance of color in the tubes.

Barfoed’s Test Result Interpretation

  • The presence of Brick red precipitate detects the presence of reducing monosaccharides in the sample.
  • If the color appears within the first few minutes, the sample contains reducing monosaccharides.
  • However, if the color appears later than the first 3 minutes, the sample is of reducing disaccharides.

Precautions

  • A briskly boiling water bath should be used for obtaining reliable results.
  • Freshly prepared reagent must be used.

Applications

  • Barfoed’s test is used for distinguishing monosaccharides from reducing disaccharides.

Limitations

  1. This test cannot be used to detect sugar in urine as urine contains Cl– ions, which might interfere with the reaction.
  2. If a higher concentration of disaccharides is present in a sample, it might give a positive result.

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