Have you ever accidentally spilled water on a handwritten note and watched the ink separate into a rainbow of different colours? This happens due to chromatography, a physical separation technique. It works by causing a distribution of substances between two different phases.
The mobile phase is the substance that moves, which in this case is the liquid solvent (like water or ethanol). The stationary phase is the material that does not move, which is the chromatography paper.
Separation occurs because different substances in a mixture have different affinities for each phase. The distance a substance travels depends on its solubility in the mobile phase and its attraction to the stationary phase. A substance that is highly soluble in the solvent and less attracted to the paper will spend more time in the mobile phase, travelling further up the paper.
To create a successful chromatogram, the experimental setup must be carefully controlled. The starting baseline (origin) must be drawn in pencil, not pen, because pencil graphite is completely insoluble and will not dissolve into the solvent.
The solvent level in the beaker must sit below the pencil line so the sample spots do not wash directly into the liquid reservoir. A lid is placed over the beaker to reduce evaporation and ensure the internal atmosphere is saturated with solvent vapour.
Sample spots must be kept small (about 2-3 mm) to prevent them from smudging into each other. If you are separating colourless substances, such as amino acids, you must use locating agents or UV light to make the spots visible after the experiment.
A pure substance is easily identified on a chromatogram because it will produce a single spot in all solvents. By contrast, an impure substance (a mixture) will separate into two or more distinct spots.
If a substance does not move from the baseline at all, it is insoluble in that specific solvent.
To identify unknown components in a mixture, you can compare them to a known reference sample run on the exact same paper. If a spot in the unknown mixture matches the exact vertical position of the reference spot, they are likely the same substance. Because different chemicals can sometimes travel the same distance in one solvent, you should confirm the identity by repeating the test with a different solvent.
To identify substances quantitatively, we calculate their value. This is a ratio comparing the distance the substance travelled to the distance the solvent front travelled.
values are simply a ratio of two lengths, meaning they have no units and always fall between and . Once calculated, you can compare your value against a standard database of known values (for that specific solvent and temperature) to identify the substance.
Worked Example:
Calculate the value for a spot that travels while the solvent travels .
Step 1: Measure the distance from the pencil baseline to the centre of the spot. (Given as ). Always measure from the baseline, never the bottom of the paper.
Step 2: Measure the distance from the pencil baseline to the solvent front. (Given as ).
Step 3: Substitute the values into the formula.
Step 4: Calculate the final answer and round appropriately (usually to 2 decimal places).
Students often think values can be larger than 1; if your answer is greater than 1, you have accidentally divided the solvent distance by the spot distance.
In explanation questions, AQA examiners actively look for the phrase 'distribution between phases' to award marks for how chromatography separates mixtures.
When measuring the distance moved by a substance, always measure from the pencil baseline to the exact centre of the spot to ensure accuracy.
If an exam question asks why the starting line is drawn in pencil, explicitly state that pencil graphite is 'insoluble' rather than just saying 'it won't run'.
Mobile phase
The phase in which molecules can move; in paper chromatography, this is the liquid solvent.
Stationary phase
The phase in which molecules cannot move; in paper chromatography, this is the chromatography paper.
Chromatogram
The visual output (the paper with separated spots) produced after the chromatography process is complete.
Baseline (origin)
The starting pencil line where sample spots are applied before running the chromatogram.
Locating agents
Chemicals or techniques (like UV light) used to make colourless substances visible on a completed chromatogram.
Pure substance
A single element or compound that is not mixed with any other substance, producing only one spot on a chromatogram.
Reference sample
A known pure substance run alongside unknown mixtures to identify components by comparing their vertical positions.
Solvent front
The furthest point reached by the solvent as it moves up the stationary phase.
Put your knowledge into practice — try past paper questions for Chemistry
Mobile phase
The phase in which molecules can move; in paper chromatography, this is the liquid solvent.
Stationary phase
The phase in which molecules cannot move; in paper chromatography, this is the chromatography paper.
Chromatogram
The visual output (the paper with separated spots) produced after the chromatography process is complete.
Baseline (origin)
The starting pencil line where sample spots are applied before running the chromatogram.
Locating agents
Chemicals or techniques (like UV light) used to make colourless substances visible on a completed chromatogram.
Pure substance
A single element or compound that is not mixed with any other substance, producing only one spot on a chromatogram.
Reference sample
A known pure substance run alongside unknown mixtures to identify components by comparing their vertical positions.
Solvent front
The furthest point reached by the solvent as it moves up the stationary phase.