Qualitative Analysis of unknown organic compound for detection of carbohydrate

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Experiment No. 5

Objective

Qualitative Analysis of unknown organic compound for detection of carbohydrate

References:

  1. Dr. Jain K.S., Dr. Miniyar P.B., A practical book of Pharmaceutical organic chemistry , Nirali publication, edition 4th, page No. 1.31-1.33
  2. Vogel Arthere, Elementary practical organic chemistry, 2nd edition, 1966, published by CBS publisher, page No. 89

Requirement

Glasswares and Instrument

  1. Test tubes
  2. Pipette or dropper
  3. Beaker (for water bath)
  4. Test tube holder
  5. Bunsen burner or hot water bath

Chemicals

  1. Molisch Reagent (α-naphthol in ethanol)
  2. Concentrated sulfuric acid (H₂SO₄)
  3. Fehlind Sol A (Copper(II) sulfate solution) & B (Alkaline solution of potassium sodium tartrate (Rochelle salt) in sodium hydroxide)
  4. Benedict Reagent ((contains copper(II) sulfate, sodium carbonate, and sodium citrate))
  5. Sample (glucose, fructose, sucrose)
  6. Distilled water

Theory:

Carbohydrates are organic compounds containing carbon, hydrogen, and oxygen. They include simple sugars (monosaccharides), disaccharides, and polysaccharides. Molisch’s test is commonly used as a general test. This test is based on the dehydration of carbohydrates by concentrated sulfuric acid to form furfural or hydroxymethylfurfural, which then react with α-naphthol (Molisch’s reagent) to produce a purple or violet ring at the interface of the acid and the aqueous layer.

In Fehling’s Test: Reducing sugars react with the copper ions in Fehling’s reagent to form copper(I) oxide (Cu2O), which precipitates out as a brick-red solid which indicates the presence of reducing sugars. 
In Benedict’s Test:  Similar to Fehling’s test, reducing sugars reduce the copper(II) ions in Benedict’s reagent to copper(I) oxide, which precipitates out. The reagent changes color, ranging from green, yellow, orange, or red, depending on the concentration of reducing sugars. 
 
Procedure
Molisch Test
  1. Take 2 mL of the aqueous carbohydrate solution in a clean test tube.
  2. Add 2–3 drops of Molisch’s reagent (α-naphthol in ethanol) to the solution.
  3. Carefully add 1–2 mL of concentrated sulfuric acid along the sides of the test tube using a dropper or pipette. Do not mix. Allow the acid to settle at the bottom.
  4. Observe the formation of a violet or purple ring at the junction of the two liquids.

D-glucose+con H2SO4——>5 Hydroxy methyl furfural—>+alpha nephthol—>purple/violet ring

Fehling’s Test:

  1. Mix 1 mL of Fehling’s solution A and 1 mL of Fehling’s solution B in a test tube to prepare fresh Fehling’s reagent.
  2. Add 2 mL of the carbohydrate solution (or a few crystals of the solid sample dissolved in water) to the test tube.
  3. Gently heat the mixture in a boiling water bath for 2–5 minutes or directly over a low flame with a test tube holder.
  4. Observe any color change or precipitate (red) formation.

Benedict Test

  1. Take 2 mL of the carbohydrate solution in a clean test tube.
  2. Add 2 mL of Benedict’s reagent to the test tube.
  3. Mix well and heat the solution in a boiling water bath for 3–5 minutes, or gently over a flame.
  4. Observe the color change or precipitate formation.

  5. The degree of color change can give a rough estimate of sugar concentration:

    • Blue (no change): No reducing sugar

    • Green: Trace amount

    • Yellow: Low concentration

    • Orange: Moderate concentration

    • Brick-red: High concentration

Observation 

Molisch Test: ………………………….color is found

Fehling Test: ………………………….color is found

Benedict Test:  ………………………….color is found

Result: Qualitative analysis of organic compound for detection of carbohydrate was done successfully.

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