Rf values and thin-layer chromatography: how to calculate and read them
How thin layer chromatography (TLC) works, how to calculate an Rf value step by step with a labelled plate, how spots are seen, and why Rf values change.
By Zachery Casey · Published · Reviewed · Editorial policy
An Rf value is the distance a spot travels divided by the distance the solvent front travels, both measured from the starting line. It always lies between 0 and 1. Rf values come from thin-layer chromatography (TLC), one of the quickest ways to see how many substances are in a sample: a drop of the sample goes on a coated plate, a solvent climbs the plate, and the substances separate into spots. This guide explains how a TLC plate is run, how to calculate an Rf value, how spots are made visible, and what TLC can and can't tell you.
For the general idea of stationary and mobile phases, see how chromatography works. Terms are defined in the glossary.
What is TLC?
TLC is a form of chromatography done on a flat plate instead of in a column. The plate is the stationary phase: a thin coating of a fine powder, usually silica gel, sometimes alumina. The mobile phase is a solvent or solvent mixture that soaks up the plate by capillary action, the same way water creeps up a paper towel.
As the solvent rises, it carries the substances in the sample with it. Each one sticks to the coating to a different extent. Those that stick less travel further. Those that stick more stay near the start. The result is a column of separate spots.
Silica is polar, so on a silica plate polar substances tend to hold back and less polar ones travel further. Changing to a more polar solvent moves everything further up the plate. For why some molecules are more polar than others, see chemical bonds and polarity.
How a TLC plate is run
- Draw a starting line. A light pencil line about 1 cm from the bottom marks where the samples go. Pencil is used because ink would dissolve and run.
- Spot the samples. A tiny amount of each dissolved sample is touched onto the line with a fine glass capillary. Small, tight spots give sharp results. A known reference substance is often spotted alongside for comparison.
- Develop the plate. The plate stands in a covered jar with a shallow pool of solvent below the starting line. The jar is covered so the air inside fills with solvent vapour. That stops the solvent evaporating from the plate as it rises (Chemguide).
- Mark the solvent front. When the solvent is near the top, the plate is taken out and the solvent's highest point is marked straight away, before it dries and the line disappears.
- Make the spots visible. Most substances are colourless, so the plate is viewed under UV light or treated with a stain (see below).
What is an Rf value?
The Rf value (retention factor) is a number that describes how far a substance travels up a TLC plate or chromatography paper compared with the solvent. A substance that sticks strongly to the plate has a low Rf value; one that travels almost with the solvent has an Rf close to 1.
How to calculate an Rf value
The Rf value compares how far a spot moved with how far the solvent moved:
Rf = distance moved by the spot ÷ distance moved by the solvent front
Both distances are measured from the starting line. Measure to the centre of each spot.
Worked example
A plate is developed until the solvent front is 7.5 cm above the starting line. Three spots appear:
| Spot | Distance from starting line | Rf |
|---|---|---|
| A | 1.8 cm | 1.8 ÷ 7.5 = 0.24 |
| B | 3.6 cm | 3.6 ÷ 7.5 = 0.48 |
| C | 5.4 cm | 5.4 ÷ 7.5 = 0.72 |
Spot A held onto the plate most strongly and spot C the least. An Rf value always lies between 0 and 1 and has no units. A spot that doesn't move from the line has an Rf of 0. A spot that runs with the solvent front has an Rf close to 1, which means the solvent was too strong to separate it.
How spots are made visible
| Method | How it works | Notes |
|---|---|---|
| UV light (254 nm) | The plate's fluorescent coating glows; compounds that absorb UV block it and show as dark spots | Doesn't harm the spots. Best for compounds with rings or alternating double bonds |
| Iodine vapour | Iodine forms coloured complexes with many organic compounds, giving yellow-brown spots | The spots fade as the iodine evaporates |
| Ninhydrin | Reacts with free amino groups, such as those in amino acids, to give coloured spots | Destructive. Used for amino acids and some peptides |
| Other chemical stains | React with particular groups to give coloured spots | Destructive, so the plate can't be reused |
UV and iodine details are from LibreTexts, and ninhydrin from Chemguide.
Why Rf values change
An Rf value belongs to a substance under one set of conditions, not to the substance alone. It shifts when any of these change:
- The solvent. A small change in the mixture, such as a little more of the polar part, moves every spot.
- The plate. Different brands, coatings and thicknesses hold substances differently.
- Temperature. Warmer conditions change how strongly substances stick and how fast the solvent rises.
- Saturation of the jar. An uncovered or freshly filled jar lets solvent evaporate from the plate, which distorts the run.
- How much sample was spotted. Overloaded spots smear into streaks, which makes the centre hard to judge.
So published Rf values are only a rough guide. The reliable way to compare two substances is to run them side by side on the same plate.
What TLC can and can't tell you
TLC is quick, cheap and needs very little sample. It is widely used to follow a reaction as it happens, check fractions during purification, or see roughly how many components a sample has.
It has clear limits:
- It isn't quantitative. The size and darkness of a spot give only a rough idea of how much is there.
- It can miss things. A substance that doesn't absorb UV and doesn't react with the stain stays invisible.
- Matching Rf values doesn't prove identity. Two different substances can travel the same distance.
- Closely related molecules often run together. A peptide and an impurity that differs by one residue may give a single spot.
That is why a peptide purity figure comes from HPLC, which separates far more finely and measures each peak's size, and identity comes from mass spectrometry. For reading the trace HPLC produces, see reading a chromatogram.
Related guides
For the main types of chromatography side by side, see how chromatography works. For how peptides are purified on a larger scale, see how peptides are purified. For which solvents mix with which, see the solvent miscibility and polarity chart. An independent lab tests every lot we sell for purity by HPLC and identity by mass spectrometry. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.
More on how the tests work
- How HPLC works: separation, retention time and what it can't tell youWhat is HPLC? How an HPLC system separates a sample, with a labelled diagram, HPLC vs UHPLC, normal vs reversed phase, and what retention time means.
- Light and the electromagnetic spectrum: wavelength, UV and absorbanceThe electromagnetic spectrum in order, from radio waves to gamma rays: how wavelength, frequency and energy link, and why peptides are measured with UV light.
- Reading a chromatogram: peaks, baseline, signal-to-noise and calibration curvesWhat a chromatogram is and how to read one: retention time, peak area, signal-to-noise ratio and detection limits, plus how a calibration curve works.