1. Benedict’s Test for Reducing Sugars

• Principle: Detects reducing sugars that can donate electrons to Benedict’s reagent (copper(II) sulphate).
• Method:

1. 1. Add Benedict’s reagent (blue) to the sample.
   2. Heat in a water bath at 80°C for 5 minutes.
   3. Positive result: Colour change from blue → green → yellow → orange → brick red (depending on concentration of reducing sugar)​.
   4. This colour change is seen as a red precipitate forms when a reducing sugar is present.

• Benedict’s solution contains blue copper(II) sulphate ions (Cu²⁺).
• When heated, the reducing sugar donates electrons to Cu²⁺, reducing it to copper(I) oxide (Cu₂O).

Formation of a the Red Precipitate:

• Cu₂O is insoluble in water and appears as a brick-red precipitate.
• The colour change depends on the concentration of the reducing sugar:
  • Blue (no sugar)
  • Green (trace amounts)
  • Yellow (low concentration)
  • Orange (moderate concentration)
  • Brick-red (high concentration)

The Benedict's test is considered semi-quantitative because it provides an approximate indication of the concentration of reducing sugars in a sample, rather than an exact numerical value.

The estimation is subjective as it depends on visual interpretation of colour, which can vary between observers.

To quantify the concentration of reducing sugar present you could:

• Filter the precipitate and dry it
• Weigh the precipitate
• The greater the mass of precipitate, the greater the concentration of reducing sugar.

This is a fully quantitative measure and is objective. Meaning it will be more accurate.

2. Benedict’s Test for Non-Reducing Sugars

• Principle: Some disaccharides (e.g., sucrose) do not react with Benedict’s reagent unless first hydrolysed into monosaccharides.
• Method:

1. 1. Perform the Benedict’s test; if the result remains blue, proceed with the next steps.
   2. Add dilute hydrochloric acid (HCl) and heat to hydrolyse glycosidic bonds.
   3. Neutralise with sodium hydrogen carbonate (NaHCO₃) which is alkaline..
   4. Repeat the Benedict’s test.
   5. Positive result: Colour change from blue → brick red​.

3. Emulsion Test for Lipids

• Principle: Lipids dissolve in ethanol but not in water, forming an emulsion.
• Method:

1. 1. Add ethanol to the sample and shake well.
   2. Pour the mixture into a test tube containing water.
   3. Positive result: A white, milky emulsion forms​.

4. Iodine Test for Starch

• Principle: Iodine binds to the helical structure of starch, producing a colour change.
• Method:

1. 1. Add iodine solution (iodine dissolved in potassium iodide) to the sample.
   2. Positive result: Colour change from orange-brown → blue-black​.

5. Biuret Test for Proteins

• Principle: The copper(II) ions in Biuret reagent react with peptide bonds in proteins.
• Method:

1. 1. Add Biuret reagent (contains sodium hydroxide and copper(II) sulfate) to the sample.
   2. Positive result: Colour change from light blue → lilac/purple​.