Thin Layer Chromatography (TLC) – Principle, procedure
What is Chromatography?
Chromatography means separation of mixture or compounds. The components travel through a stationary phase at different speeds and get separated. The mixture is dissolved in mobile phase and is carried through stationary phase.
Thin Layer chromatography is a form of planar chromatography. The stationary phase is TLC plate (silica gel) or alumina. The plate is run through a solvent which is known as the mobile phase.
The principle of TLC is where mixture components are separated between a fixed stationary phase and a liquid mobile phase by differential affinities between the two phases. The separation is through adsorption.
Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance.
What is High Performance Thin Layer Chromatography?
HPTLC (High Performance Thin Layer Chromatography) is an advanced version of TLC. TLC is qualitative whereas HPTLC is qualitative and quantitative. HPTLC works on similar principles as TLC but the practice is fully automated and GLP/USP/EP compliant. As a liquid chromatography technique, HPTLC can analyse most non-volatile organic samples with ease.
TLC is performed manually. Samples are applied on TLC plates. The plate (stationary phase) is silica gel backed by Alumina or glass. The plate is run through a solvent (Mobile phase) system for separation of the samples.
1. Preparation of sample and plate for the experiment. Cut the TLC plate in 10×10, 20×10 dimensions. Mark the plate with a pencil at 70mm.
2. Application of sample on TLC plate. TLC involves manual spotting whereas HPTLC has automatic sampler for e.g., CAMAG ATS 4. You can spot the samples on the plate. Mark the sample application area with pencil and start the spotting process.
3. Development of plate. The plate is kept in a solvent system such that the samples are not immersed in the mobile phase. For this purpose, use CAMAG TTC (Twin through chambers). Observe the plate while it is developed and remove the plate.
4. After development is finished. Air dry the plate and observe the separated compounds. Only the compounds observed in white light can be seen by naked eye. For UV active and fluorescent compounds, we need a UV chamber or CAMAG Visualizer.
5.Analysis of results requires interaction with detected compounds. CAMAG TLC-MS coupling uses Mass Spectrometry for the verification of the chemical structure of analytes. Analytes can be directly sent to an MS or the results can be collected for further analysis offline.