What the Rf value is
In thin-layer chromatography (TLC) and paper chromatography, a mixture is spotted on a baseline and a solvent climbs the plate by capillary action. Each compound travels a different distance, depending on how strongly it sticks to the stationary phase compared with how well it dissolves in the solvent. The retention factor (Rf) expresses that distance as a fraction.
The formula
Rf = distance traveled by the spot ÷ distance traveled by the solvent front
Both distances are measured from the baseline: to the center of the spot, and to the line the solvent reached when you removed the plate. Rf is always between 0 and 1 and has no unit.
Example
The solvent front reached 8.0 cm. Spot 1 is centered at 3.2 cm and spot 2 at 4.0 cm:
- Rf₁ = 3.2 ÷ 8.0 = 0.40;
- Rf₂ = 4.0 ÷ 8.0 = 0.50;
- with spot widths of 0.4 and 0.5 cm, the resolution is 2 × 0.8 ÷ 0.9 ≈ 1.8: the spots are well separated.
Resolution
Rs = 2 × (distance between spot centers) ÷ (sum of spot widths)
- Rs ≥ 1.5: spots fully separated;
- Rs between 1 and 1.5: separated but nearly touching;
- Rs below 1: overlapping spots.
Getting useful Rf values
- Aim for Rf values between 0.2 and 0.8. Spots near the baseline or the front are poorly separated and their Rf is unreliable.
- If spots stay too low, use a more polar eluent (on silica); if they run too high, use a less polar one.
- Mark the solvent front with a pencil as soon as you remove the plate, before it evaporates.
- Keep the baseline above the solvent level in the tank, or the spots dissolve into the solvent.
Using Rf to identify compounds
Rf depends on the stationary phase, the solvent, the temperature, the plate thickness and the saturation of the chamber. To compare a sample with a reference, run them side by side on the same plate, ideally with a co-spot (sample and reference applied together). The same Rf suggests, but does not prove, the same compound.
Visualizing spots
Colorless compounds can be seen under UV light (254 nm) on plates with a fluorescent indicator, or revealed with iodine vapor, ninhydrin for amino acids, or permanganate and other stains.