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12.2 Analysing Chromatograms

GCSE Chemistry · AQA · Chapter 12: Chemical Analysis

A free GCSE Chemistry lesson for AQA (8462) from OBA School: how chromatography separates substances, calculating Rf values and using chromatograms to identify substances. Watch the video, read the explanation and worked examples, then try the revision questions. You can work through the full lesson, with every video, practice quizzes and printable worksheets with mark schemes, in our free online course.

What you will learn

  • Explain how paper chromatography separates substances using a stationary phase and a mobile phase
  • Calculate Rf values from a chromatogram, and rearrange the equation
  • Use Rf values and reference substances to identify the components of a mixture
  • Explain how a chromatogram shows whether a substance is pure

Key words

  • Chromatography: A method for separating the substances in a mixture so that they can be identified.
  • Stationary phase: The phase that does not move. In paper chromatography it is the paper.
  • Mobile phase: The phase that moves through the stationary phase. In paper chromatography it is the solvent.
  • Chromatogram: The pattern of spots produced by chromatography.
  • Solvent front: The furthest point the solvent reaches up the paper.
  • Locating agent: A chemical sprayed on a dried chromatogram to make colourless substances show up as coloured spots.
  • Rf value: Distance moved by a substance ÷ distance moved by the solvent. It has no units.

How paper chromatography works

Small spots of the samples are put on a pencil baseline. The bottom of the paper is dipped into a solvent, which soaks up the paper and carries the substances with it.

Each substance is shared (distributed) between the two phases. A substance that is more soluble in the solvent (mobile phase) and less attracted to the paper (stationary phase) spends more time moving, so it travels further up the paper. Different substances move different distances, so the mixture separates.

Key fact: Thin-layer chromatography (TLC) works in the same way. Its stationary phase is a thin layer of a solid such as silica on a glass or plastic plate.

Calculating Rf values

Rf = distance moved by substance ÷ distance moved by solvent

Measure both distances from the baseline: to the centre of the spot, and to the solvent front. Rf is always less than 1 and has no units.

Worked example: Rf of the blue dye

blue spot moved 5.2 cm; solvent moved 8.0 cm
Rf = 5.2 ÷ 8.0 = 0.65
The red spot moved 2.0 cm, so its Rf = 2.0 ÷ 8.0 = 0.25.

Answer: Rf (blue) = 0.65

Worked example: rearranging

A dye has Rf = 0.40. The solvent moves 7.5 cm. How far does the dye move?
distance moved by dye = Rf × distance moved by solvent = 0.40 × 7.5 = 3.0 cm

Answer: 3.0 cm

Identifying substances

  • A given compound always has the same Rf value in the same solvent (with the same paper and conditions).
  • Compare the spots of an unknown with reference substances run on the same paper: spots at the same height (same Rf) are likely to be the same substance.
  • The Rf value of a compound changes if you change the solvent, so you must compare Rf values measured with the same solvent.
  • If two substances have the same Rf in one solvent, run the chromatogram again with a different solvent to tell them apart.
  • A substance that does not dissolve in the solvent stays on the baseline.
  • dye X; Rf in water 0.36; Rf in ethanol 0.81
  • dye Y; Rf in water 0.36; Rf in ethanol 0.52
  • unknown; Rf in water 0.36; Rf in ethanol 0.52

In water, X and Y have the same Rf, so we cannot tell which the unknown is. In ethanol, the unknown matches dye Y.

Comparing with a database, and colourless substances

Chemists do not need to run every possible reference substance. Instead they measure the Rf value of each spot and look it up in a database of Rf values. Because Rf is a ratio, it does not matter how long the paper is or how far the solvent was allowed to run.

Remember: An Rf value depends on the solvent and the temperature (and the type of paper). A database value can only be used if the chromatogram was run with the same solvent at the same temperature. Change either one and the balance of attractions to the two phases changes, so the spot moves a different distance.

Many substances, such as amino acids and sugars, are colourless. After the chromatogram has dried it is sprayed with a locating agent, a chemical that reacts with the substances to form coloured spots. The spots can then be measured in the usual way.

Pure or mixture?

A pure compound gives one spot in every solvent. A mixture usually gives two or more spots. If a sample gives a single spot in one solvent, it might still be a mixture whose components happen to have the same Rf, so test it in another solvent too.

Exam tip: In practical questions: the baseline is drawn in pencil because graphite is insoluble and will not run; the solvent must be below the baseline or the spots dissolve into the solvent in the beaker; a lid stops the solvent evaporating.

Common mistakes

  • Measuring from the bottom of the paper instead of from the baseline.
  • Measuring to the top edge of a spot instead of its centre.
  • Giving an Rf value bigger than 1, or with units. Check you divided the smaller number by the larger one.
  • Saying the paper is the mobile phase. The paper stays still; the solvent moves.
  • Saying one spot in one solvent proves a substance is pure. It needs one spot in every solvent tried.

Revision questions

Try these, then check your answers below each one.

Question 1. In paper chromatography, what is the stationary phase? (Choose from: The paper, The solvent, The pencil line, The beaker.)

Answer: The paper

Working: The paper is the stationary phase; the solvent is the mobile phase.

Question 2. In a chromatogram, a spot moves 3.6 cm from the baseline and the solvent moves 8.0 cm. Calculate the R_f value.

Answer: 0.45

Working: R_f = 3.6 ÷ 8.0 = 0.45

Question 3. True or false? An R_f value can be greater than 1.

Answer: False

Working: A substance cannot travel further than the solvent front, so R_f is always less than 1.

Related lessons

Continue in the full course

This lesson is section 12.2 (AGCCH1202) of our free GCSE Chemistry course for AQA. In the full course you get every video for this section, a self-marking practice quiz, a printable worksheet with a mark scheme, chapter tests, and your progress saved. Go to the full GCSE Chemistry course.

 

 

 

 

 

 

 

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