Paper chromatography: theory and practice

Chemical analysis • Purity, formulations, and chromatography

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What is the formula for Rf value?

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Rf = distance travelled by substance ÷ distance travelled by solvent front.

Key concepts

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Definition and basic principle

Paper chromatography is a separation technique that uses paper as the stationary phase and a liquid solvent as the mobile phase. A small spot of the mixture is placed on a pencil baseline; the solvent moves up the paper by capillary action and carries components at different rates. Different components separation causes discrete spots at different heights on the paper, forming a chromatogram. Separation occurs because each component has a different balance between solubility in the mobile phase and adsorption to the stationary phase. A stronger tendency to dissolve in the solvent causes greater migration; a stronger tendency to adhere to the paper causes less migration.

Stationary and mobile phases

The stationary phase is the paper, which holds water in its fibres and provides sites for adsorption of solute molecules. The mobile phase is the solvent or solvent mixture that moves through the paper and dissolves components. The interplay between adsorption to the paper and solubility in the solvent determines the migration distance of each solute. Changing the solvent polarity changes solute solubility and therefore relative migration. A more polar solvent increases movement of polar solutes; a less polar solvent favours movement of non-polar solutes. Paper type and moisture content alter adsorption strength, affecting separation quality.

Mechanism: cause → effect

Different solutes have different solubilities in the chosen solvent; higher solubility causes faster travel with the solvent front, producing larger migration distances. Different solutes also show different adsorption strengths to cellulose fibres in paper; stronger adsorption causes slower migration. The resulting chromatogram shows spots separated by their different migration distances. Capillary action causes solvent movement up the paper; solvent front movement carries dissolved solutes. Differences in polarity and intermolecular attractions cause selective partitioning between the mobile and stationary phases, producing separation.

Rf value: definition and interpretation

Rf (retention factor) is the ratio of the distance travelled by a substance to the distance travelled by the solvent front, measured from the baseline. Rf = (distance from baseline to centre of solute spot) / (distance from baseline to solvent front). Rf is unitless and ranges from 0 to 1. Rf values allow comparison between runs under identical conditions. Identical Rf values suggest the same substance; different Rf values indicate different substances. Rf values vary with solvent, paper, temperature and spotting technique, so comparisons require consistent experimental conditions.

Practical procedure

A pencil baseline is drawn above the solvent level and small spots of sample and standards are applied to the baseline. The paper is suspended so that only the baseline area touches the solvent. The solvent rises until it nearly reaches the top, then the paper is removed and the solvent front is marked immediately with a pencil. Spots are visualised by visible colour, UV light, or chemical locating agents. Accurate spotting, small volume, and careful timing improve resolution. Standards run alongside samples enable identification by matching Rf values. Drying and marking the solvent front immediately prevent measurement errors due to evaporation.

Limitations and factors affecting accuracy

Rf values change with solvent composition, temperature, paper type, humidity, spot size and concentration. Overloaded or large spots cause tailing and poor resolution. Poorly chosen solvent produces little separation or co-migration of components. Visual detection limits identification to coloured or detectable compounds; invisible compounds require locating reagents or UV detection. Reproducible identification requires strict control of experimental variables and running known standards under the same conditions. Rf comparison is suggestive but not definitive; further analysis may be necessary for absolute identification.

Key notes

Important points to keep in mind

Always draw the baseline in pencil to avoid ink running.

Spot volumes must be small to prevent spot spreading and tailing.

Measure distances from the pencil baseline to the centre of spots and to the solvent front.

Mark the solvent front immediately after removing the paper to avoid evaporation errors.

Run known standards under identical conditions for reliable identification.

Keep solvent depth below the baseline so spots do not dissolve directly into the solvent.

Use an appropriate solvent polarity to maximise separation of target substances.

Control temperature and humidity to improve reproducibility.

Use locating reagents or UV light for invisible compounds.

Report Rf values as unitless numbers with experimental conditions noted.