Carbohydrate Reactions: Qualitative Tests

Posted on Apr 4, 2025 in Chemistry

1. Molisch Test

Concentrated sulfuric acid (H2SO4) dehydrates monosaccharides, forming furfural (from pentoses) or 5-hydroxymethylfurfural (from hexoses). These aldehydes condense with α-naphthol to produce a purple ring. This test is positive for all free or combined carbohydrates (mono-, oligo-, and polysaccharides).

Reaction: Carbohydrate + α-naphthol + H2SO4 → Purple product (Molisch)

Reagents:

  • Concentrated sulfuric acid
  • Molisch reagent: 5g α-naphthol in 100mL ethanol

Procedure:

  • Tube A: 2mL of 1% glucose
  • Tube B: 2mL of 1% sucrose
  • Tube C: 2mL of 1% starch
  • Tube D: 2mL of distilled water
  • Add 2 drops of Molisch reagent to each tube and mix.
  • Carefully add 2mL of concentrated sulfuric acid down the side of each tube.
  • A positive reaction is indicated by a purple ring at the interface.

2. Seliwanoff Test

This test differentiates aldoses and ketoses. Ketoses react with HCl to form furfural derivatives, which condense with resorcinol to produce a red complex. Aldoses may give a faint positive reaction after prolonged heating due to epimerization to fructose.

Reagents:

  • 1% solutions of fructose (+, ketose), sucrose (+), and glucose (−, aldose)
  • Seliwanoff reagent: 0.5g resorcinol in 100mL ethanol
  • 8.4N HCl

Procedure:

  • Tube A: 1mL of 1% fructose
  • Tube B: 1mL of 1% sucrose
  • Tube C: 1mL of 1% glucose
  • Add 3mL of dilute HCl and 2mL of Seliwanoff reagent to each tube.
  • Heat in a boiling water bath and note the time until a red color appears (positive reaction).

3. Bial’s Test

This test is specific for pentoses. Pentoses heated with concentrated HCl produce furfural, which condenses with orcinol. In the presence of ferric ions, the product turns blue or blue-green.

Reagents:

  • Concentrated HCl
  • 1% solutions of xylose, glucose, ribose, and starch
  • Bial’s reagent: 1.5g orcinol + 500mL concentrated HCl + 30 drops of 10% ferric chloride

Procedure:

  • Tube A: 1mL of 1% xylose or ribose
  • Tube B: 1mL of 1% glucose
  • Tube C: 1mL of 1% starch
  • Add 10 drops of Bial’s reagent to each tube, mix, and heat in a boiling water bath.
  • A green color indicates a positive reaction.

4. Iodine Test

This test detects starch, which forms a blue complex with iodine in solution. This complex dissociates upon heating and reforms upon cooling.

Reagents:

  • 1% starch solution
  • 1% glucose solution
  • Lugol’s solution (iodine)

Procedure:

  • Tube A: 1mL of 1% glucose + 1 drop of Lugol’s solution
  • Tube B: 1mL of 1% starch + 1 drop of Lugol’s solution
  • Observe the color change. Heat in a boiling water bath and observe. Cool the tube under running water and observe.