Chromatography and Rf values

What you'll learn

Chromatography is a separation technique tested consistently across CIE IGCSE Chemistry papers, particularly in Paper 3 and Paper 6 practical assessments. This guide covers the principles of paper chromatography, the calculation and interpretation of Rf values, experimental procedures, and how to analyse chromatograms. Understanding this topic is essential for both theory papers and practical examinations.

Key terms and definitions

Chromatography — a technique used to separate mixtures into their individual components based on differences in their distribution between a mobile phase and a stationary phase.

Mobile phase — the solvent that moves through the stationary phase, carrying the components of the mixture with it (in paper chromatography, this is typically water or ethanol).

Stationary phase — the material that does not move and through which the mobile phase travels (in paper chromatography, this is the chromatography paper made of cellulose fibres).

Chromatogram — the visual result of a chromatography experiment showing the separated components as spots or bands at different positions.

Rf value (Retention factor) — a numerical value that represents the ratio of the distance travelled by a substance to the distance travelled by the solvent front, calculated as: Rf = distance travelled by substance ÷ distance travelled by solvent.

Solvent front — the furthest point reached by the mobile phase (solvent) on the chromatography paper during the experiment.

Baseline — the pencil line drawn near the bottom of the chromatography paper where the sample is applied before the experiment begins.

Locating agent — a chemical substance used to make colourless separated components visible on a chromatogram (for example, ninhydrin spray for amino acids).

Core concepts

The principle of paper chromatography

Paper chromatography separates substances based on their relative solubility in the mobile phase and their relative attraction to the stationary phase. Different substances have different solubilities and different affinities for the paper, causing them to move at different rates.

When the mobile phase travels up the chromatography paper by capillary action, it dissolves the components in the mixture. Substances that are more soluble in the solvent and less attracted to the paper travel further up the paper. Substances that are less soluble in the solvent or more strongly attracted to the paper travel shorter distances.

The separation occurs because:

• Each component has a different balance between its attraction to the stationary phase and its solubility in the mobile phase
• More soluble components spend more time dissolved in the mobile phase and travel further
• Less soluble components spend more time adsorbed to the stationary phase and travel shorter distances

Setting up a paper chromatography experiment

The correct experimental setup is frequently tested in CIE IGCSE Chemistry practical papers:

1. Draw the baseline: Use a pencil (never pen, as ink would dissolve and interfere) to draw a horizontal line approximately 1-2 cm from the bottom of the chromatography paper

2. Apply the sample: Using a capillary tube or fine pipette, place a small concentrated spot of the mixture on the baseline. Allow it to dry, then reapply to build up concentration if needed

3. Prepare the solvent: Pour the solvent into a beaker to a depth of approximately 0.5 cm — the solvent level must be below the baseline where the sample spots are placed

4. Position the paper: Place the chromatography paper vertically in the beaker, ensuring the baseline is above the solvent level. The paper should not touch the sides of the beaker

5. Cover the beaker: Place a lid or watch glass on top to create a saturated atmosphere and prevent solvent evaporation

6. Allow development: Leave undisturbed until the solvent front has travelled near the top of the paper (typically 3/4 of the way up)

7. Mark the solvent front: Remove the paper and immediately mark the position of the solvent front with a pencil before it evaporates

8. Dry the chromatogram: Allow the paper to dry completely, applying a locating agent if the separated components are colourless

Understanding and calculating Rf values

The Rf value is a characteristic property of a substance under specific chromatography conditions (same solvent, same temperature, same type of paper). It always has a value between 0 and 1 (or can be expressed as a percentage between 0% and 100%).

Formula: Rf = distance travelled by component ÷ distance travelled by solvent front

Important measurement rules for CIE IGCSE examinations:

• Always measure from the baseline (not from the bottom of the paper)
• Measure to the centre of each spot
• Measure the solvent front from the baseline to where the solvent stopped
• Use the same units for both measurements (typically cm or mm)
• Record Rf values to two decimal places

Properties of Rf values:

• An Rf value is constant for a specific substance under identical conditions
• Different substances have different Rf values under the same conditions
• Rf values can be used to identify unknown substances by comparing with known values
• If conditions change (different solvent, temperature, or paper), the Rf value changes
• A substance that does not move has an Rf of 0
• A substance that travels with the solvent front has an Rf close to 1

Interpreting chromatograms

Chromatograms provide qualitative information about mixtures:

Number of components: Each separate spot represents a different component in the mixture. A pure substance produces only one spot, while a mixture produces multiple spots.

Identity of components: Unknown substances can be identified by:

• Comparing Rf values with those of known reference substances
• Running reference samples alongside the unknown on the same chromatogram
• Matching spots that travel the same distance under identical conditions

Comparison of mixtures: Two substances are the same if:

• They produce spots at the same height on the chromatogram
• They have the same Rf value under identical conditions
• They show the same colour (for coloured substances)

Purity testing: A substance is pure if it produces only a single spot. Multiple spots indicate the presence of impurities or that the sample is a mixture.

Applications in CIE IGCSE Chemistry

Chromatography appears in various contexts across the syllabus:

Food analysis: Separating and identifying food colourings (artificial dyes like tartrazine, sunset yellow) and additives in processed foods and drinks

Forensic science: Comparing inks from different pens to determine if the same pen wrote different documents, or analysing dyes from fibres found at crime scenes

Drug testing: Identifying components in medicines or detecting banned substances in sports testing

Environmental monitoring: Detecting pollutants or pesticide residues in water samples

Quality control: Checking the purity of pharmaceutical products or ensuring food products contain the stated ingredients

Two-way chromatography

For complex mixtures where spots might overlap using one-dimensional chromatography, two-way chromatography (also called two-dimensional chromatography) provides better separation:

1. Run the chromatogram in one direction with one solvent
2. Rotate the dried chromatogram 90°
3. Run it again in the perpendicular direction using a different solvent
4. Components separate in two dimensions, reducing overlap

This technique is less commonly examined at IGCSE level but demonstrates understanding of how chromatography can be adapted.

Worked examples

Example 1: Calculating Rf values

Question: A student performed paper chromatography to separate the dyes in a food colouring. The solvent front travelled 8.0 cm from the baseline. Three spots appeared at distances of 2.4 cm, 5.6 cm, and 7.2 cm from the baseline.

Calculate the Rf value for each component. [3 marks]

Solution:

Component 1: Rf = 2.4 ÷ 8.0 = 0.30 ✓

Component 2: Rf = 5.6 ÷ 8.0 = 0.70 ✓

Component 3: Rf = 7.2 ÷ 8.0 = 0.90 ✓

Examiner tip: Show your working clearly. Each correct Rf value calculated using the formula gains the mark. Express answers to 2 decimal places unless told otherwise.

Example 2: Identifying substances using Rf values

Question: A forensic scientist analysed ink from a forged document using paper chromatography in ethanol. One component had an Rf value of 0.45. The table shows Rf values of known dyes in ethanol:

Dye	Rf value in ethanol
Methyl orange	0.65
Methylene blue	0.45
Bromothymol blue	0.32
Phenolphthalein	0.78

(a) Identify the dye present in the ink. [1 mark]

(b) Explain why pencil, not pen, should be used to draw the baseline. [1 mark]

Solution:

(a) Methylene blue ✓

(Rf value matches the component from the forged document)

(b) Pencil/graphite is insoluble in the solvent / does not dissolve ✓

(If pen was used, the ink would dissolve and move up the paper, interfering with the results)

Example 3: Practical planning

Question: A student wants to investigate whether a green food colouring is a pure substance or a mixture.

(a) Describe how the student could use paper chromatography to investigate this. [4 marks]

(b) The chromatogram shows two spots: one yellow and one blue. What does this indicate about the green food colouring? [1 mark]

Solution:

(a)

• Draw a baseline in pencil near the bottom of chromatography paper ✓
• Place a spot of the green food colouring on the baseline ✓
• Place the paper in a beaker containing solvent with the level below the baseline ✓
• Allow the solvent to travel up the paper, then mark the solvent front ✓

(Alternative valid points: cover the beaker, allow the paper to dry, measure distances travelled)

(b) The green food colouring is a mixture (of yellow and blue dyes) ✓

Common mistakes and how to avoid them

• Mistake: Measuring distances from the bottom of the paper instead of from the baseline. Correction: Always measure both the component distance and solvent front distance from the baseline where you applied the sample. The bottom of the paper is irrelevant.

• Mistake: Calculating Rf as distance travelled by solvent ÷ distance travelled by component (the fraction inverted). Correction: Remember Rf = component distance ÷ solvent distance. The component distance is always the numerator. Since components never travel further than the solvent, Rf is always less than or equal to 1.

• Mistake: Using a pen to draw the baseline or mark positions on the chromatography paper. Correction: Always use pencil. Pen ink would dissolve in the solvent and travel up the paper, contaminating your results and making the experiment invalid.

• Mistake: Allowing the initial solvent level to be above the baseline where samples are spotted. Correction: The solvent level must be below the spots on the baseline. If spots are submerged, the components will dissolve directly into the solvent instead of separating as the solvent travels upward.

• Mistake: Forgetting to mark the solvent front immediately after removing the paper. Correction: The solvent front evaporates quickly once removed from the beaker. Mark its position with pencil immediately, or your Rf calculations will be impossible.

• Mistake: Assuming that substances with the same Rf value in one solvent system are definitely identical. Correction: While matching Rf values suggest substances might be the same, confirmation requires testing in different solvent systems. Different substances can occasionally have similar Rf values in one particular solvent.

Exam technique for "Chromatography and Rf values"

• Calculation questions: Command words like "calculate" require you to show the Rf formula, substitute values, and give the answer to 2 decimal places. Always write Rf = distance of component ÷ distance of solvent front, then show your numbers. Units cancel, so Rf has no units. A correct answer without working typically earns only 1 mark when 2 are available.

• Practical descriptions: When "describe" or "outline" appears, structure your answer as numbered steps in chronological order. Questions worth 4-5 marks expect 4-5 distinct practical steps. Include: drawing baseline in pencil, applying sample, solvent level below baseline, covering container, allowing solvent to rise, marking solvent front.

• Explanation questions: For "explain" command words (often 2-3 marks), state what happens AND why. For example, explaining why substances separate requires mentioning different solubilities AND different attractions to paper, linking these to different distances travelled.

• Paper 6 skills: Practical papers may require you to measure distances on actual chromatograms or photographs. Use a ruler to measure to the centre of spots in mm, then convert to cm if appropriate. Show all measurements and calculations clearly in the designated spaces.

Quick revision summary

Chromatography separates mixtures using a mobile phase (solvent) moving through a stationary phase (paper). Components separate based on different solubilities and attractions to the paper. The Rf value = distance travelled by component ÷ distance travelled by solvent front, calculated from the baseline to spot centres. Each substance has a characteristic Rf value under specific conditions. Pure substances give one spot; mixtures give multiple spots. Always use pencil for the baseline, ensure solvent level is below baseline spots, and mark the solvent front immediately after removing the paper. Rf values identify unknowns by comparison with reference data.