Thin Layer Chromatography (TLC): Principle and Procedure
TLC or Thin Layer Chromatography
TLC is a type of planar chromatography.
It is routinely used by researchers in the field of phytochemicals, biochemistry, and so forth, to identify the components in a compound mixture, like alkaloids, phospholipids, and amino acids.
It is a semi-quantitative method consisting of analysis.
High-performance thin-layer chromatography (HPTLC) is the more sophisticated or more precise quantitative version.
Similar to other chromatographic methods, thin layer chromatography is also based on the principle of separation.
- The separation depends on the relative affinity of compounds towards stationary and the mobile phase.
- The compounds under the influence of the mobile phase (driven by capillary action) travel over the surface of the stationary phase. During this movement, the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus, the separation of components in the mixture is achieved.
- Once separation occurs, the individual components are visualized as spots at a different level of travel on the plate. Their nature or character are identified using suitable detection techniques.
TLC system components consist of
- TLC plates, preferably ready-made with a stationary phase: These are stable and chemically inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The stationary phase on the plates is of uniform thickness and is in fine particle size.
- TLC chamber. This is used for the development of the TLC plate. The chamber maintains a stable environment inside for proper development of spots. It also prevents the evaporation of solvents and keeps the process dust-free.
- Mobile phase. This comprises of a solvent or solvent mixture. The mobile phase used should be particulate-free and of the highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, a stationary phase.
- A filter paper. This is moistened in the mobile phase, to be placed inside the chamber. This helps develop a uniform rise in a mobile phase over the length of the stationary phase.
The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize. These days, however, ready-made plates are preferred.
- With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots.
- Then, samples solutions are applied on the spots marked on the line in equal distances.
- The mobile phase is poured into the TLC chamber to a leveled few centimeters above the chamber bottom. A moistened filter paper in the mobile phase is placed on the inner wall of the chamber to maintain equal humidity (and also thereby avoids edge effect this way).
- Now, the plate prepared with sample spotting is placed in the TLC chamber so that the side of the plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid.
- The plate is then immersed, such that the sample spots are well above the level of mobile phase (but not immersed in the solvent — as shown in the picture) for development.
- Allow sufficient time for the development of spots. Then remove the plates and allow them to dry. The sample spots can now be seen in a suitable UV light chamber or any other methods as recommended for the said sample.
- It is a simple process with short development time.
- It helps with the visualization of separated compound spots easily.
- The method helps to identify the individual compounds.
- It helps in isolating of most of the compounds.
- The separation process is faster and the selectivity for compounds is higher (even small differences in chemistry is enough for clear separation).
- The purity standards of the given sample can be assessed easily.
- It is a cheaper chromatographic technique.
- To check the purity of the given samples.
- Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics, and more.
- To evaluate the reaction process by assessment of intermediates, reaction course, and so forth.
- To purify samples, i.e., for the purification process.
- To keep a check on the performance of other separation processes.
Being a semi-quantitative technique, TLC is used more for rapid qualitative measurements than for quantitative purposes. But due to its rapidity of results, easy handling, and inexpensive procedure, it finds its application as one of the most widely used chromatography techniques.