Paper Chromatography Detailed

Paper chromatography is tested on Paper 6 (Alternative to Practical) and in theory questions on separation techniques. The 0620 syllabus requires candidates to describe the method, calculate Rf values, and interpret chromatograms.

Principle

Chromatography separates mixtures of dissolved substances based on differences in their solubility in the solvent and their attraction to the paper. Substances that are more soluble in the solvent travel further up the paper. Substances that are more attracted to the paper fibres (cellulose) travel less far.

Apparatus and method

Equipment needed

• Chromatography paper (or filter paper)
• Pencil (not pen — ink would dissolve and interfere)
• Ruler
• Capillary tube or fine glass rod (for spotting)
• Suitable solvent (water for water-soluble substances; ethanol or other organic solvents for others)
• Beaker or chromatography tank with a lid
• Pin or paper clip to suspend the paper

Step-by-step procedure

1. Draw a pencil line (the baseline or origin) about 2 cm from the bottom of the chromatography paper
2. Using a capillary tube, place small spots of the samples on the pencil line, evenly spaced. Label them in pencil below the line
3. Pour a shallow layer of solvent into the beaker — the solvent level must be below the pencil line
4. Carefully lower the paper into the beaker so the bottom edge dips into the solvent, but the spots remain above the solvent
5. Cover the beaker with a lid to prevent the solvent evaporating
6. Allow the solvent to rise up the paper by capillary action
7. When the solvent front is near the top, remove the paper and immediately mark the position of the solvent front with a pencil line
8. Allow the paper to dry
9. If the substances are colourless, use a locating agent (UV light, iodine vapour, or ninhydrin spray) to reveal the spots

Calculating Rf values

Rf = distance moved by substance / distance moved by solvent front

Both distances are measured from the origin (baseline) to the centre of the spot (for the substance) or to the solvent front line.

Worked example

A chromatogram shows:

• Solvent front moved 10.0 cm from the origin
• Substance A moved 7.5 cm
• Substance B moved 3.0 cm

Rf of A = 7.5 / 10.0 = 0.75

Rf of B = 3.0 / 10.0 = 0.30

Properties of Rf values

• Always between 0 and 1 (a substance cannot travel further than the solvent)
• Characteristic for a given substance under specific conditions (same solvent, same temperature, same paper)
• If conditions change, the Rf value changes — you must compare spots run under identical conditions
• No units

Interpreting chromatograms

Identifying pure and impure substances

• A pure substance produces a single spot on the chromatogram
• An impure substance (mixture) produces two or more spots

Identifying components of a mixture

Run reference (known) substances alongside the unknown mixture on the same paper. If a spot in the mixture has the same Rf value as a reference spot, it is likely the same substance. This is more reliable than comparing with Rf values from a different experiment because conditions may differ.

Example interpretation

If a food colouring produces three spots at Rf 0.25, 0.55, and 0.80, and reference dyes give these values:

• Dye X: Rf 0.25
• Dye Y: Rf 0.55
• Dye Z: Rf 0.80
• Dye W: Rf 0.40

Then the food colouring contains dyes X, Y, and Z but not W.

Why use pencil, not pen?

Pen ink dissolves in the solvent and would be carried up the paper along with the samples, contaminating the chromatogram. Pencil graphite is insoluble in common chromatography solvents, so the baseline and labels stay in place.

Applications at IGCSE level

• Identifying which dyes are present in a food colouring
• Checking whether a substance is pure (one spot) or a mixture (multiple spots)
• Matching unknown substances to known reference samples
• Identifying amino acids in a protein hydrolysis (using ninhydrin spray)

Worked exam question

A student carries out paper chromatography on an unknown ink. The solvent front moves 8.0 cm from the baseline. The ink separates into two spots at 2.4 cm and 5.6 cm from the baseline. (a) Calculate the Rf values of the two components. [2] (b) What does this tell you about the ink? [1] (c) A known dye has an Rf value of 0.30. Is this dye present in the ink? [1]

(a) Rf₁ = 2.4 / 8.0 = 0.30 [1]; Rf₂ = 5.6 / 8.0 = 0.70 [1]

(b) The ink is a mixture of (at least) two substances [1]

(c) Yes — the first component has Rf = 0.30, which matches the known dye (assuming they were run under the same conditions) [1]

Common exam mistakes

1. Measuring from the bottom of the paper instead of from the baseline/origin — all distances must be measured from the pencil line.
2. Drawing the baseline with pen — pen ink dissolves and runs with the solvent.
3. Saying a substance is “definitely the same” because it has the same Rf — without running them side by side, you can only say they are “likely” the same.
4. Allowing the solvent level to be above the baseline — the spots wash off and no separation occurs.