Thin Layer Chromatography
Introduction to Thin Layer Chromatography (TLC)
Thin Layer Chromatography (TLC), is a quick and easy qualitative technique for monitoring the course of a reaction or comparing a sample against authentic material. TLC is a chromatographic technique which separates components based on their affinity for the stationary phase and their mobility in the mobile phase.
TLC Plates
A TLC plate consists of a material mounted on a support giving a thin, uniform layer which is called the stationery phase. Silica gel is the most common stationary phase, providing a normal phase, polar plate. Other material may be used, such as long silica gels with long alkyl-chains giving a reversed phase plate, or non-silica based materials such as alumina. Binders are often included, along with fluorophores to enable visualisation under UV light.
The support is usually glass, aluminium foil or plastic which the stationary phase is applied to in a thin layer and is typically baked to bond the stationary phase to the plate. There are many different commercially available TLC plates produced by a variety of manufacturers. The main plates used in teaching labs are:
Manufacturer
Merck
Part number
1.05554.0001
Backing
Aluminium foil
Stationary phase material
Silica Gel 60
Stationary phase thickness
200 µm
Fluorophore wavelength
254 nm
Commercial TLC plates are usually sold as large sheets which are cut down to useable sizes in the lab. There is a compromise between having plates sufficiently large to give good separation and small enough to be quick to run and cost effective. The exact dimensions will depend on the purpose of the TLC, for example spotting column fractions might be more efficient if carried out on wide plates. A plate roughly 4 cm by 7 cm gives a fairly general purpose size and can be efficiently cut from 20 cm by 20 cm plates which are a common commercial size.
Uses of TLC
Thin layer chromatography is a simple and quick qualitative technique to examine the components of a mixture. It can be used to check the purity of materials, compare substances or to monitor reactions. Step by step instructions are provided for these three uses in the tabs on this page.
Resources required
Thin layer chromatography requires simple, readily available equipment in addition to TLC plates. Typical equipment would consist of:
TLC Plate
A TLC plate suitable for the compounds to be analysed.
Solutions of analyte
Samples to be analysed usually need to be in solution. A good solvent should readily dissolve the compound and be sufficiently volatile to evaporate from the plate leaving only the substance to be analysed behind. Reaction mixtures can often be spotted directly onto plates.
Pencil and ruler
To mark a baseline onto the TLC plate, typically 1 cm above the bottom of the plate. It is important to use pencil as inks will dissolve in the solvent and travel up the plate.
TLC tank
A TLC tank for developing the TLC plate. Usually a 250 mL beaker and watch glass fulfil this purpose, but other glassware can be used (eg glass jars with lids or commercial tanks). A filter paper is usually added to saturate the atmosphere inside the tank.
Measuring cylinder
To measure out the solvent system for running the TLC. The solvent depth usually wants to be approximately 0.5 cm in the tank. For a 250 mL beaker this usually equates to 10 mL of solvent.
Solvent system
A system suitable for the TLC plate and compounds of interest. The solvent system may need to be experimentally determined. For normal phase chromatography, polar compounds are retained by the plate and need a more polar solvent system in order for compounds to travel further on the plate. A good solvent system is one that gives clear separation for the compounds of interest.
TLC spotter
A thin glass capillary is usually used to apply small amounts of a solution of the analyte to the TLC plate. Spotters can be obtained from commercial sources or home-made.
UV lamp
Compounds on the TLC plate can be analysed by examining the plate under UV-light. Most TLC plates contain a fluorescent chromophore which fluoresces in the visible region when placed in UV light. Compounds on the plate quench this fluorescence and show as dark spots. UV lamps are used to check loading of plates prior to running the TLC and also to identify components after running.
TLC stains
TLC plates can be stained in dips which react with the chemicals on the TLC plate. For more information see the section on visualising plates.
TLC and column chromatography
Whilst TLC can be used as a discrete technique, it is often used as the first step in preparing a compound for purification using flash column chromatography. The two techniques are very closely related, where TLC is qualitative and flash column chromatography a preparative purification technique.