A Brief Note on Thin Layer Chromatography

About the Study

TLC stands for Thin-Layer Chromatography, which is a chromatographic technique for separating non-volatile substances. Thin-layer chromatography uses an inert substrate, such as glass, plastic, or aluminum foil, coated with a thin layer of adsorbent material, commonly silica gel, aluminum oxide (alumina), or cellulose. The chromatographic point is referred to as the solid phase.

A solvent or alcohol combination (known as the mobile phase) is drawn up the plate via capillary action after the sample has been put to the plate. Different analytes ascend the TLC plate at various rates, leading to separation. The stationary phase and the mobile phase have different properties. Non-polar mobile phases, such as heptane, are utilized with silica gel, which is a relatively polar material. Chemists can fine-tune the bulk characteristics of the mobile phase by using a mixture as the mobile phase.

The spots are displayed after the experiment. The sheets are often coated with a phosphor, and black spots form on the sheet where chemicals absorb the light impinging on a specific location. Spots can also be visualised through chemical processes; anisaldehyde, for example, creates colourful adducts with a variety of compounds, while sulfuric acid chars most organic molecules, leaving a dark spot on the sheet.

To quantify the results, the Retardation Factor (RF), also known as the retention factor, is calculated by dividing the distance travelled by the material under consideration by the total distance travelled by the mobile phase. The solvent absorption must be halted before the mobile phase reaches the end of the stationary phase for the result to be quantitative. In general, a material with a structure similar to the stationary phase would have a low RF, whereas one with a structure similar to the mobile phase will have a high Rf. Retardation variables are common, however they might vary based on the mobile and stationary phases' specific conditions. As a result, scientists frequently apply a sample of a known compound alongside the unknown compounds to the sheet.

Thin-layer chromatography can be used to track a reaction's progress, identify components in a given mixture, and determine a substance's purity. Assessing ceramides and fatty acids, detecting pesticides or insecticides in food and water, analysing the dye content of fibres in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identifying medicinal plants and their constituents are all examples of these applications.
The original method can be improved in a variety of ways, including automating the various procedures, increasing the resolution gained using TLC, and allowing for more precise quantitative analysis. HPTLC, or "High-Performance TLC," is the name given to this approach. HPTLC often employs thinner stationary phase layers and lower sample volumes, resulting in less resolution loss due to diffusion. The separation is built on the mobile and stationary phases being partitioned differently. Differences in the partition coefficient of such a compound cause differential retention on the stationary phase, therefore, were impacting separations.

Thin layer chromatography procedure

TLC plates: This is where the thin layer of the stationary phase is applied. In nature, they are inert or steady. For better analysis, the layer of stationary phase is kept consistent across these plates. People who conduct tests frequently prefer ready-to-use plates.

Mobile phase: A solvent is used in the mobile phase (or solvent mixture). The solvent used must be chemically inert, of the highest purity feasible, and free of particulates. The TLC spots will only be able to develop after that.

TLC chamber: It keeps dust particles out of the process and prevents the solvent from evaporating. Inside this chamber, a consistent atmosphere is maintained in order to properly develop the spots.

The TLC procedure's separation principle is based on a compound's relative affinity for the mobile and stationary phases. The process starts with the mobile phase travelling over the surface of the stationary phase. The higher affinity compounds gain less speed during this movement than the lower affinity compounds. As a result, they are separated.

After the process is completed, various spots at different levels can be found on the stationary surface, reflecting separate parts of the mixture. In principle, compounds that are more attracted to the stationary phase maintain their place at lower levels on the surface, while others rise to higher levels. As a result, their locations may be observed correspondingly.

A thin coating of absorbent substances, such as aluminium oxide, silica gel, or cellulose, is used for the latter. An active substrate is a sheet of plastic, glass, or aluminium foil that has been coated with this layer. In the TLC process, the mobile phase is a solvent or a combination of solvents.