Thin Layer Chromatography (TLC)

General procedure

The below procedures apply to all TLC, whether for the purposes of comparing compounds or for monitoring a reaction.


Preparing plates

To prepare a TLC plate for loading compounds a baseline needs to be drawn onto the plate to ensure that all compounds run from the same point on the plate. In pencil, draw a feint line (base line) approximately 1 cm from the bottom of one end of the TLC plate.  Avoid scratching the silica off the plate with your pencil as this will affect the chromatography.  On the base line, mark application points for the samples which will be applied to the plate.


Loading samples

Loading samples onto a TLC plate successfully takes some practice. The ideal application leaves a tight spot on the plate that can easily be visualised under UV-light. TLC spotters made from glass are the most common applicators. These want to have a small diameter to enable the application of small quantities of material. Compounds should be applied as solutions a drop at a time by touching the TLC spotter to the application point on the TLC plate. Ideally the plate is only touched briefly to minimise the amount of solution being drawn out of the spotter. The spot should ideally only be a couple of mm in diameter. The plate can be examined under a UV lamp to view the intensity of the spot. Weak spots can have further amounts of compound added by spotting again on top of the spot.


TLC Tank

A TLC tank will need preparing for use. This may consist of a commercial TLC tank, a screw top jar or most commonly a 250 mL beaker and watch glass. The tank should have a suitable size filter paper added to the tank. This is to try and saturate the tank with solvent vapour which is important to minimise the evaporation of solvent from the TLC plate as it runs (and would affect the Rf value of analytes). A suitable solvent mixture would then be added to the beaker, filling it to around 0.5 cm depth. For a 250 mL beaker, around 10 mL is usually about the right amount of mobile phase to add. Ideally the TLC tank is prepared slightly ahead of time to enable the filter paper to draw up the solvent and fill the tank with solvent vapour giving a saturated atmosphere. TLC tanks can be used to run a number of plates, however it is important to remember that solvent compositions may change over time due to differing evaporating rates from solvent mixtures and may need to be regularly remade.


Running a plate

TLC plates are placed into the TLC tank with baseline and application points at the bottom of the plate. Tweezers are usually used to hold the plate to add or remove plates from the tank. It is important that the application points sit above the level of the solvent in the tank, as otherwise the compounds will diffuse into the solution and affect the chromatography by contaminating the plate. The plate should be sloped at an angle with the backing of the top of the plate resting against the filter paper in the TLC tank. The watch glass should be quickly replaced and the plate allowed to develop. The mobile phase will travel up the plate gradually and should do so in as straight a line as possible. Once the mobile phase reaches close to the top (1-2 cm away), the TLC should be removed (again using tweezers) and the solvent front marked as quickly as possible with pencil as the solvent will rapidly evaporate from the plate.


Analysing plates

Plates are usually analysed initially under UV light and any spots observed are marked by circling around their outline with a pencil. Sometimes UV light is sufficient analysis, however TLC stains are routinely used to aid analysis. Further information can be found under visualising plates.


Determining retention factors

Retention factor (Rf) is the relationship between the distance the solvent front and the compound spot on the TLC plate. It is defined as:

formula showing that retention factor R F is equal to the distance moved by the spot of interest divided by the distance moved by the solvent front

Compounds that have travelled most of the way up the plate will have an Rf value close to 1, whereas compounds close to the baseline have an Rf value close to 0.


The Rf value for a compound travelling on a particular type of TLC plate in a given solvent system should be reproducible. As a result Rf values are often quoted in experimental information for compounds, quoting the solvent system used along with information on the TLC plate and visualisation methods.

Diagraming showing a T L C plate, this is annotated to show the baseline and solvent front as well as the compound spot that has moved half way up the plate and the original application point.

Checking purity of a material

TLC can be used to quickly check if a compound consists of a single component. Whilst this method has largely been surpassed by other analytical methods (for example NMR spectroscopy), it can still be a useful tool. Typically the TLC plate consists of a single application point. A small amount of the compound of interest is added to a sample vial and dissolved in a suitable solvent and applied to the plate. After the plate has been run, it is visualised and hopefully only a single spot is observed. The presence of multiple spots could indicate that the material is impure, or that the material is unstable on the stationary phase of the TLC plate. Where known, the Rf can be compared to known values.


Comparing a sample with authentic material

Comparing a compound prepared in the laboratory with a known sample is usually acheived using three application points on a TLC plate. The left channel has authentic material applied to it, the right channel the sample of interest. In the middle, it is common to spot both authentic material and the sample of interest, giving what is termed a co-spot. The co-spot can help show where compounds are actually different, event though the Rf values are similar. The plate on the left shows the sample to be the same, whereas the plate on the right has two different, closely running compounds. The co-spot is clearly enlarged and can be seen to consist of two components. Without a co-spot this plate could easily have resulted in concluding a positive match by TLC.

Diagram showing 2 T L C plates. Both plates have 3 sample spots, the left spot is authentic material, the right spot is the sample of interest and the middle spot is a co-spot. One T L C plate shows that when the sample and authentic material are the same, the finished T L C will show 3 equal spots at the same R F value. The other T L C plate shows that when the sample and authentic material are not the same, the finished T L C will show the co-spot as 2 overlapping but not identical spots.

Monitoring a reaction

Reaction monitoring is the most common use for TLC. For most reactions three channels are used, from left to right:



However, sometimes further reagents are spotted onto plates (eg non-limiting reagents or known products) in order to aid identification. Example TLCs are shown below. On the left a typical plate where a reaction is part way through, and on the right a reaction showing complete conversion.

Diagram showing 2 T L C plates. Both plates have 3 sample spots, the left spot is the limiting reagent, the right spot is the reaction mixture and the middle spot is a co-spot. One T L C plate shows that when the reaction is not yet complete, the finished T L C will show 1 limiting reagent spot, 2 co-spots spots and 2 reaction mixture spots. The other T L C plate shows that when the reaction is complete, the finished T L C will no longer show a reaction mixture spot at the same R F value as the limiting reagent.Diagram showing 2 T L C plates. Both plates have 3 sample spots, the left spot is the limiting reagent, the right spot is the reaction mixture and the middle spot is a co-spot. One T L C plate shows that when the reaction is not yet complete, the finished T L C will show 1 limiting reagent spot, 2 co-spots spots and 2 reaction mixture spots. The other T L C plate shows that when the reaction is complete, the finished T L C will no longer show a reaction mixture spot at the same R F value as the limiting reagent.