8.1.3Chemical Analysis

Chromatography — AQA GCSE Combined Science Trilogy 8464 - Chemistry Revision Notes & Topical Questions

5 min read 12 parts Lesson 4 of 9

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Part 1 of 10Principle of paper chromatography

Use
- separates substances with different solubilities in a solvent, such as the dyes in black ink.
Spots
- a pencil line is drawn and spots of sample placed on it.
- Pencil
  - used because ink would run with the samples.
- Solvent
  - the paper is lowered into solvent with the pencil line above the surface.
- Movement
  - the solvent rises by capillary action, carrying substances with it.
- Separation
  - more soluble substances travel further.

Terms
- paper chromatography is the technique and a chromatogram is the paper showing the result.
Same substance
- gives the same pattern and identical spots.
- Reference spot
  - a known compound used for comparison with unknown spots.
- Mixture
  - separates into several spots, one for each component.
- Pure substance
  - gives one spot.
- Impure sample
  - shows more than one spot.

Problem
- colourless substances such as amino acids and sugars cannot be seen on the paper.
Locating agent
- a chemical that reacts with the sample to give a coloured product.
- Use
  - apply after the run so the spots become visible to the naked eye.

Definition
- the distance moved by a substance divided by the distance moved by the solvent.
Equation
- Rf = distance travelled by substance ÷ distance travelled by solvent.
  What the symbols mean: R_f has no unit and is always between 0 and 1 · both distances are measured from the baseline
- Units
  - none, as it is a ratio.
- Always between 0 and 1
  - a substance cannot move further than the solvent.
- Constant
  - the Rf of a compound is the same under the same conditions.
- Identification
  - compare the Rf of an unknown with known values.

Measurement tip
- always measure from the pencil line, not from the bottom of the paper.

Small spots
- large spots overlap and separate poorly.
Solvent level
- keep it below the baseline.
- Lid
  - cover the container so solvent does not evaporate from the paper.
- Stop
  - remove the paper before the solvent front reaches the top so the distance can be measured.
- Drying
  - dry in air before using a locating agent.

Observation	Conclusion
Spot level with a reference spot	Sample contains the reference substance
One spot only	Sample is probably pure
Several spots	Sample is a mixture
Spot not matching any reference	Unknown substance present

Rf use
- compare calculated Rf values with a data table for the same solvent.

Food
- testing whether a colouring is permitted by comparing with known dyes.
Forensics
- matching an ink or dye from a sample to a suspect source.
- Medicine
  - detecting drugs and checking that a product is pure.
- Colourless samples
  - amino acids from a protein can be separated and sprayed with a locating agent.

Explain why the starting line is drawn in pencil. [2]2 marks
1. Pencil does not dissolve in the solvent.
2. Ink would run up the paper with the samples.
A spot travels 4.2 cm while the solvent travels 6.0 cm. Calculate the Rf. [2]2 marks
1. Rf = 4.2 ÷ 6.0.
2. = 0.70.
A chromatogram of a food dye shows two spots. What does this show? [1]1 mark
1. The dye is a mixture of at least two substances.
Why must a locating agent be used for amino acids? [2]2 marks
1. Amino acids are colourless.
2. The agent reacts to give a coloured product that can be seen.

Term	Meaning
Chromatography	A method to separate substances based on their different solubilities.
Chromatogram	The paper showing the separated spots.
Solvent front	The furthest point reached by the solvent.
Locating agent	A reagent that makes colourless spots visible.
Rf value	Distance moved by a substance divided by distance moved by solvent.

Last updated: 20 October 2026