Chromatography Experiment

Aim

To separate and identify the dyes present in a sample of ink or food colouring using paper chromatography, and to calculate Rf values for each separated component.

Apparatus

• Chromatography paper (or filter paper)
• Pencil and ruler
• Capillary tubes (or fine glass pipettes)
• Beaker (250 cm3) with a cover (watch glass, cling film, or foil lid)
• Solvent: distilled water (for water-soluble dyes) or ethanol (for dyes less soluble in water)
• Samples: unknown ink or food colouring
• Reference dyes: known dyes for comparison (e.g., pure food colourings)
• Ruler (for measuring distances)

Method

1. Cut a piece of chromatography paper to fit inside the beaker, tall enough to reach near the top.
2. Using a pencil and ruler, draw a straight baseline approximately 2 cm from the bottom of the paper.
3. Using a capillary tube, place a small, concentrated spot of the unknown sample on the baseline. Apply the spot carefully, keeping it as small as possible (a large spot gives blurred results). Allow it to dry, then apply a second spot on top to increase the concentration.
4. On the same baseline, place spots of reference/known dyes at equal intervals, labelling each in pencil below the baseline.
5. Pour solvent into the beaker to a depth of approximately 1 cm — the solvent level must be below the baseline so the spots are not submerged in the liquid.
6. Carefully lower the paper into the beaker so the bottom edge dips into the solvent but the spots remain above the solvent surface.
7. Cover the beaker with a watch glass or lid to prevent the solvent evaporating.
8. Leave undisturbed until the solvent front has risen to near the top of the paper (but not over the top edge).
9. Remove the paper and immediately mark the position of the solvent front with a pencil line.
10. Allow the paper to dry in air. The chromatogram is now ready for analysis.

Expected results

Each dye travels a different distance up the paper, depending on its relative solubility in the mobile phase (solvent) and its attraction to the stationary phase (paper). A mixture produces multiple spots; a pure substance produces one spot.

For a typical black ink: the chromatogram may show separate spots of blue, red, and yellow, revealing that the black ink is a mixture of several dyes.

For food colourings: each food colouring may produce one spot (if it is a single pure dye) or several spots (if it is a mixture).

Analysis

Calculating Rf values

For each spot on the chromatogram, measure:

• The distance from the baseline to the centre of the spot (d)
• The distance from the baseline to the solvent front (D)

Rf = d / D

The Rf value is always between 0 and 1, and has no units. Each substance has a characteristic Rf value in a given solvent under specific conditions (temperature, type of paper).

Identifying unknowns

Compare the Rf values of the unknown spots with the Rf values of the known reference dyes. If a spot from the unknown has the same Rf as a reference dye (and is at the same height on the same chromatogram), they are likely the same substance.

If no reference dyes are available, compare calculated Rf values with published data — but these are only valid if the same solvent, paper type, and temperature were used.

Sources of error

• Spots too large: Large spots spread and overlap, making individual dyes difficult to distinguish. Apply small, concentrated dots.
• Solvent above baseline: If the baseline spots are submerged in the solvent, the dyes dissolve directly into the solvent pool and do not separate properly.
• Lid not used: Solvent evaporation from the surface of the paper changes the solvent front position and gives inaccurate Rf values. Always cover the beaker.
• Paper moved during development: Disturbing the paper while the solvent is rising disrupts the separation. Leave the beaker completely still.
• Measuring errors: Always measure from the centre of each spot to the baseline, not from the edge. Use a ruler and measure to the nearest millimetre.

Exam tips

Examiners award separate marks for each key detail: pencil baseline (not pen), spots above the solvent level, lid on the beaker, and marking the solvent front before drying. State all four when describing the method.

When interpreting a chromatogram, count the spots to determine how many components are in the mixture. If asked whether a substance is pure, look at how many spots it produces — one spot indicates a pure substance (or at least a single component detectable by this method); multiple spots indicate a mixture.

Rf value calculations are straightforward but require careful measurement. Show your working: Rf = distance moved by substance / distance moved by solvent front. Round to two decimal places. If the question gives you data, check units and ensure both distances are measured from the same starting point (the baseline).

For supplement-level answers, explain the separation in terms of the two phases: substances more soluble in the mobile phase travel further; substances more attracted to the stationary phase stay closer to the baseline. Each substance partitions differently between the phases, giving different Rf values.