Qualitative Analysis of Carbohydrates

This section explains how to detect unknown samples that are carbohydrates, lipids, or proteins
Qualitative tests determine the presence or absence of a substance, without measuring its quantity.

Carbohydrates are classified based on the number of sugar units
- Monosaccharides: Simple sugars with one sugar unit (e.g., glucose, fructose, mannose, galactose)
- Disaccharides: Two sugar units (e.g., lactose, maltose, sucrose)
- Oligosaccharides: Contain 3-10 sugar units
- Polysaccharides: Contain more than 10 sugar units

Aim: Detects the presence of carbohydrates in a sample.
Reagents:
- α-naphthol
- Concentrated sulfuric acid
Procedure:
- Add a few drops of α-naphthol to 1 ml of the test solution in a test tube.
- Carefully add 1 ml of concentrated sulfuric acid to the test tube, allowing it to flow down the side of the tube.
- Mix well.
Result:
- Positive: A purple to violet ring appears at the junction of the acid and solution. This indicates the presence of carbohydrates.
- Negative: No ring appears, suggesting the sample might be a lipid or protein.
Principle:
- The concentrated sulfuric acid hydrolyzes carbohydrates into simpler forms.
- Monosaccharides undergo dehydration, producing a 5-carbon cyclic compound:
  - Furfural for pentoses
  - Hydroxymethyl furfural for hexoses
- α-naphthol reacts with these products, forming a purple ring.

Aim: Distinguishes between reducing and non-reducing sugars, and also between mono/disaccharides and polysaccharides.
Reagents:
- Benedict's reagent containing:
  - Sodium carbonate (alkaline conditions)
  - Sodium citrate (complexes with copper ions to prevent deterioration)
  - Copper(II) sulfate pentahydrate
Procedure:
- Add 1 ml of Benedict's reagent to the test solution and boil for 5 minutes.
Result:
- Positive: Formation of a red, green, or yellow precipitate indicates a reducing sugar (e.g., glucose) is present.
- Negative: The solution remains clear or slightly blue, indicating no reducing sugar is present.
Principle:
- Reducing sugars contain free aldehyde or ketone groups, which can reduce copper(II) ions in Benedict's reagent to copper(I) ions.
- This reduction causes a color change from blue to red, green, or yellow, depending on the amount of reducing sugar present.
- Avoid heating for more than 5 minutes as the heat may cause hydrolysis of polysaccharides and sucrose, leading to false positives.

Aim: Distinguish reducing monosaccharides from reducing disaccharides.
Reagents:
- Barfoed's reagent containing:
  - Copper acetate
  - Diluted acetic acid
Procedure:
- Add 2 ml of Barfoed's reagent to the test solution and boil for 3 minutes.
Result:
- Positive: A brick-red precipitate forms within 1-2 minutes for monosaccharides. Disaccharides take longer (7-12 minutes) to react if a positive result is obtained.
Principle:
- Monosaccharides react faster with the Barfoed's reagent than disaccharides because they are more readily oxidized by the copper ions.

Aim: Detects the presence of ketohexoses (e.g., fructose) and distinguishes them from aldoses.
Reagents:
- 0.05% resorcinol (m-hydroxybenzene)
- Concentrated HCl
Procedure:
- Add 2 ml of Seliwanoff's reagent to the test solution and boil for 2 minutes.
Result:
- Positive: A red color develops within 2 minutes, indicating the presence of fructose.
- Negative: Aldoses, if present, may produce a faint pink to cherry red color if the test is prolonged.
Principle:
- Ketohexoses are more rapidly dehydrated than aldoses in the presence of concentrated acid.
- Fructose undergoes dehydration to yield 5-hydroxymethyl furfural.
- This dehydrated ketose reacts with resorcinol, forming a complex (not a precipitate) that shows a deep cherry red color.
- Aldoses may react slightly, but the product and reaction time help distinguish between carbohydrates.

The process of breaking down starch into simpler sugars (monosaccharides and disaccharides) requires the presence of acid (e.g., HCl) and heat. This can be tested using the Benedict's test after the hydrolysis is complete.