All Labs

Chromatography Lab

Watch compounds separate as a solvent front rises through a paper strip. Measure retention factors (Rf) for ink dyes, leaf pigments, and food colors, and learn how polarity controls how far each compound travels.

Guided Experiment: Ink Separation Investigation

Black ink appears to be a single color. What do you predict will happen when ink is separated by chromatography? Will it separate into multiple colors?

Write your hypothesis in the Lab Report panel, then click Next.

Chromatography Strip

Blue dyeRf=0.85
Red dyeRf=0.65
Yellow dyeRf=0.45
Green dyeRf=0.30

Controls

Rf Values

Run the experiment to see Rf values.

Rf Bar Chart

Data Table

(0 rows)
#TrialCompoundSolvent Front(cm)Spot Distance(cm)RfPolarity
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0 / 500
0 / 500

Reference Guide

Paper Chromatography

Paper chromatography separates mixtures by allowing a solvent to move up a paper strip by capillary action. Compounds in the mixture travel with the solvent at different rates depending on how strongly they interact with the paper (stationary phase).

In normal-phase chromatography, the stationary phase (paper) is polar. Polar compounds stick to the paper more and travel shorter distances. Non-polar compounds interact less with the paper and travel further with the solvent.

Retention Factor (Rf)

The Rf value is the ratio of how far a compound travels to how far the solvent front travels.

Rf=distance solutedistance solvent frontR_f = \frac{\text{distance solute}}{\text{distance solvent front}}

Rf values range from 0 (compound stays at origin) to 1 (compound travels with the solvent front). Each compound has a characteristic Rf value for a given solvent and stationary phase combination.

Polarity and Separation

The key rule in normal-phase chromatography:

  • High Rf (travels far) = low polarity compound
  • Low Rf (stays near origin) = high polarity compound

This is because polar compounds form stronger interactions with the polar paper (hydrogen bonds, dipole forces) and are retained more. Non-polar compounds prefer to stay dissolved in the moving solvent.

non-polar: high Rf  |  polar: low Rf

Identifying Unknowns

Rf values are reproducible under the same conditions, making them useful for identifying unknown compounds. To identify an unknown:

  1. Run unknown and known standards on the same strip
  2. Measure Rf for each spot
  3. Match unknown Rf to the closest standard
  4. A match within ±0.05 is a reliable identification

Real labs also use co-spotting: applying an unknown and a standard at the same origin point. A single merged spot after development confirms they are the same compound.