Thin layer chromatography is commonly considered as an easy, quick, and cheap technique for the separation, and tentative detection of a broad range of components. The several steps involved in thin layer chromatography. First, the sample spotted on a coated plate. Then the TLC plate is placed in a covered TLC chamber containing a small amount of a mobile phase or a solvent. As the mobile phase moves to the plate by capillary action, analytes are traveling at different rates because of their interaction with the stationary phase and mobile phase. After the development of chromatograms by achieving different spots or bands, the plate is removed and the compounds are visualized. Once the compounds are visuals the Rf value can be determined. The resolution of TLC is better than paper chromatography and equal to or less than high-performance liquid chromatography (HPLC).
The various factors that affecting the resolution, separation and Rf value in TLC are as follows.
Mobile phase composition: The suitable combination of solvents in a mobile phase is necessary to separate the components this will mainly affect the separation and resolution.
Selection and quality of solvent: As the mobile phase carries the compound up the TLC plate, the solvent selection will more impact on the separation in thin layer chromatography. Each time of analysis the high-quality solvents are needed to be used as low-quality solvents affect isolation
The polarity of the solvent or mobile phase: The solvent polarity is related to the Rf value, resolution, and separation of molecules, the difference in polarity of solvent maybe affects the result of TLC.
Solvent saturation: RF values in thin layer chromatography mainly rely on the solvent saturation of the atmosphere in the TLC chamber.
The concentration of the sample: The proper concentration of the sample has been required since a very concentrated and diluted sample cannot be properly separated.
Spotting of the TLC plate: Sampling techniques may also affect the retention factor, with the more volume of the sample resulting in a larger and a diffuse band of compounds, making it unclear to determine the distance correctly.
TLC Plate: Mostly silica or alumina is used to prepare the plate. The TLC plate can affect the retention factor, resolution, and separation of molecules since the uniformity and thickness of the absorbent layer can vary from plate to plate.
Temperature: The slight changes in the temperature of the system can influence the result, hence control of the working temperature may be needed.
Sampling technique: Every chromatographer has a different technique to apply the sample; these variations can cause to change the retention factor.
Commonly asked questions on TLC chromatography are as follows.
Why iodine is used in TLC?
The TLC plate is saturated with iodine vapor when the eluted TLC plate is put in the iodine chamber and separated spots become light brown since iodine has a high vapor pressure.
What are the advantages of thin-layer chromatography?
TLC is rapid, with multiple simultaneous samples separated. It requires a very small sample amount, it can be in micro-litre. TLC has higher Sensitivity compared to paper chromatography. Separated molecules can be seen by UV light. Silica gives better selectivity.
What are the disadvantages of TLC?
Compared to HPLC, GC, and column chromatography, TCL has a short stationary phase. Just qualitative analysis is possible. It is possible to separate only soluble components. Automation (HPTLC) makes it more expensive. The results can be affected by humidity and temperature, as it is an open system
What are the uses of thin-layer chromatography?
TLC is used to separate and determine the number of solutes present in the sample mixture. It can be used for identifying the analytes, determining their purity. The progress of a reaction can be monitored by TLC. It is broadly used in the isolation of compounds of pharmaceutical formulations.
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