Biochemical tests

Laura Armstrong

Teacher

Laura Armstrong

Recall Questions

This topic requires prior knowledge of biochemical tests from GCSE. You can test your knowledge on these below.

What is a reducing sugar?

A sugar that can donate electrons to another chemical. Examples include glucose, maltose, and lactose​.

Why do lipids form an emulsion in the emulsion test?

Lipids are insoluble in water but soluble in ethanol. When mixed with water, lipid droplets disperse, forming a white emulsion​.

What happens when proteins are present in the Biuret test?

Biuret reagent will change colour from light blue to purple​.

Topic Explainer Videos

Check out these @LauraDoesBiology videos that explain biochemical tests, or read the full notes below. Once you've gone through the whole note, try out the practice questions!

Biochemical tests

1. Benedict’s Test for Reducing Sugars

  • Principle: Detects reducing sugars that can donate electrons to Benedict’s reagent (copper(II) sulphate).
  • Method:
    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. 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. 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. 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. Add Biuret reagent (contains sodium hydroxide and copper(II) sulfate) to the sample.
    2. Positive result: Colour change from light blue → lilac/purple​.

Key Terms

  • Reducing Sugar: A sugar that donates electrons to Benedict’s reagent (e.g., glucose, maltose)​.
  • Non-Reducing Sugar: A sugar that does not react with Benedict’s unless hydrolysed first (e.g., sucrose)​.
  • Emulsion: A suspension of lipid droplets in water, appearing white and cloudy​.
  • Biuret Reagent: A solution containing sodium hydroxide and copper(II) sulfate, used to test for proteins​.
  • Semi-quantitative: A semi- quantitative test or measurement provides an approximate or a relative indication of the amount of a substance present, rather than an exact numerical value.
  • Subjective: Refers to something that is based on personal interpretations, or perspectives rather than measurable evidence.
  • Quantitative: Refers to something that is measured or expressed using numerical. 
No answer provided.

Exam Tip

When describing colour changes, always mention the original and final colour to get full marks.

In Benedict’s test, a brick red precipitate indicates high sugar concentration, while green or yellow suggests low concentration​.

No answer provided.

A scientist tested a food sample for starch, proteins, and lipids. Describe the biochemical tests used and state the expected positive results. (6 marks)

1. Starch Test (Iodine Test)

  • Add iodine solution to the sample.
  • Positive result: Colour change from orange to blue-black​.

2. Protein Test (Biuret Test)

  • Add Biuret reagent and mix.
  • Positive result: Colour change from blue to lilac/purple​.

3. Lipid Test (Emulsion Test)

  • Mix sample with ethanol, then add water.
  • Positive result: A white, cloudy emulsion forms​.

Describe how a student could carry out a chemical test for reducing sugar and suggest how he could estimate the amount of reducing sugar in the sample (the student did not have a colourimeter). (5 marks)

  • Add Benedict’s reagent
  • Heat / boil 
  • Forms precipitate / colour change from blue to, green / yellow / orange / brown / (brick) red;

Concentration estimated from EITHER 

  • Observe degree of colour change
  • Compare colour with known concentration solutions;

OR

  • Filter and weigh the precipitate
  • Greater mass/ weight = more reducing sugar present

Practice Question

Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!