In science, thin-layer chromatography (TLC) is a flexible and popular analytical method for separating and assessing mixtures of substances. It relies on the idea that mixture components migrate differently between stationary and mobile phases.
Fundamentally, Thin Layer Chromatography is a chromatographic method for identifying and separating components from mixtures. TLC uses a stationary phase that is deposited as a thin layer over a flat, inert support such as glass, plastic, or aluminium foil in contrast to other chromatographic techniques. This thin layer serves as the stationary phase and is frequently made of alumina or silica gel.
Spotted close to the TLC plate’s base is the sample combination that has been dissolved in an appropriate solvent or solvent mixture. The mixture’s ingredients are carried up the plate by the solvent as a result of capillary action. Different chemicals in the mixture partition between the stationary and mobile phases according to their different affinities as the solvent flows, producing a clear separation.
The concepts of partition chromatography and adsorption are the foundation of separation in thin-layer chromatography. Compounds stick to the stationary phase’s surface in adsorption chromatography; the strength of this adherence is dictated by the chemical characteristics of the component and the stationary phase.
Principle of Thin Layer Chromatography
The principle behind Thin Layer Chromatography (TLC) is to separate substances in a mixture according to the way they bind to the stationary and mobile phases.
Stationary Phase
- A thin layer of an adsorbent substance, such as alumina or silica gel, evenly distributed on a flat, inert substrate, such as glass, plastic, or aluminium foil, serves as the stationary phase in TLC.
- The matrix on which the separation takes place is the stationary phase. It interacts with the chemicals in the mixture through the presence of polar or charged groups.
Mobile Phase
- By capillary action, a solvent or combination of solvents passes through the stationary phase as the mobile phase.
- The kind of chemicals being separated and how they interact with the stationary phase determines which mobile phase is optimal.
Mechanism of Thin Layer Chromatography
- The mobile phase, or solvent, is allowed to ascend the TLC plate when a sample combination is put to the stationary phase close to the base of the plate.
- The sample components are carried by the mobile phase as it advances along the plate. The solvent’s capacity to interact with the stationary phase is what causes the movement, which is caused by capillary action.
- Based on their different affinities, components of the sample mixture divide between the stationary and mobile phases. greater interactions between compounds and the stationary phase cause them to travel more slowly, whereas greater interactions between compounds and the mobile phase cause them to move quicker.
- The components separate as a result of this differential partitioning as they go up the TLC plate at varying speeds.
Retention Factor (Rf)
A quantitative metric called the retention factor (Rf) is employed in TLC to describe a compound’s mobility. It is computed as the ratio of the compound’s application-point travel distance to the solvent front’s travel distance. Every chemical has a unique Rf value under certain circumstances that are determined by how it interacts with the stationary and mobile phases.
Visualization and Analysis
The TLC plate is taken out of the developing chamber and allowed to dry once it has developed and the solvent front has reached the top of the plate. Using different methods like UV light, iodine vapour, or chemical staining, the separated components show spots or bands on the plate. By calculating the distances travelled by the solvent front and each spot, the Rf values of the spots may be determined. Rf values are used to evaluate the purity of the sample and to identify and compare different chemicals.
Materials Required
- TLC plates (pre-coated with silica gel or alumina)
- Sample mixture to be analyzed
- Developing chamber (glass or plastic container with a lid)
- Mobile phase (solvent or solvent mixture)
- Capillary tubes or micropipettes for sample application
- UV lamp or other visualization techniques
- Ruler or measuring tape
Also Read| Centrifugation; An Overview of Centrifuge Structure, Principle, Types and Techniques
Procedure
- Usually, TLC plates are pre-coated with a thin layer of an adsorbent substance, such as alumina or silica gel. If not, the plates must be covered with the adsorbent substance and heated in an oven to activate it.
- Make sure there are no impurities on the TLC plates and that they are clean.
- Put the sample combination in a suitable solvent and dissolve it. The type of substances being examined determines which solvent is best.
- Make sure there are no particles in the sample solution that might clog the capillary tube while applying it. It should be transparent.
- Spot the sample solution onto the TLC plate in the vicinity of the base, or bottom, using a micropipette or capillary tube. Use tiny, uniformly spaced dots to avoid overlaps while separating.
- To ensure that the sample is distributed evenly over the TLC plate, let the spots dry fully.
- Based on the polarity of the compounds that need to be separated, select the proper developing solvent or mobile phase.
- A tiny quantity of the developing solvent should be poured into the developing chamber’s bottom, making sure the solvent level is lower than the spots on the TLC plate when the plate is within the chamber.
- Make sure the spots on the spotted TLC plate are above the solvent level when you carefully place it into the development chamber.
- To stop the solvent from evaporating and to let the plate develop, cover the developing chamber. Through capillary action, the solvent moves up the plate and picks up the components of the sample.
- As the solvent front draws closer to the top of the TLC plate, track the development of the chromatographic separation. This might take a few minutes to many hours, depending on the chemicals that need to be separated and the solvent solution.
- Take the TLC plate out of the developing chamber and let it dry fully after the solvent front has reached the top of the plate.
- Use the proper visualisation techniques, such as UV light, iodine vapour, or chemical staining, to see the separated components on the TLC plate.
- Measure the distances that each spot travels from the application point and the solvent front’s distance.
- Using the formula Rf = Distance travelled by spot / Distance travelled by solvent front, determine the retention factor (Rf) for each spot.
Also Read| HPLC – High-Performance Liquid Chromatography
Applications
1. Analysis of pharmaceuticals
In the pharmaceutical business, TLC is frequently used to analyse chemical molecules, find contaminants, and assess the purity of drugs.
Quality Control: To guarantee adherence to regulatory requirements, pharmaceutical companies regularly test raw materials, intermediates, and finished medication products using TLC.
2. Analysis of food and beverages
Food additives, preservatives, colours, and flavours in food and beverage goods are all analysed using TLC. It aids in guaranteeing adherence to rules and guidelines on food safety.
Detection of adulterants: TLC helps ensure food quality and consumer safety by helping to identify adulterants, pollutants, and microbial toxins in food and beverage samples.
3. Clinical Analysis
Hormonal analysis: In clinical research and diagnostic testing, TLC is used to analyse hormones, steroids, and other endocrine substances.
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Graduated from the University of Kerala with B.Sc. Botany and Biotechnology. M.Sc. Biotechnology from the University of Kerala. Attended certificate course in Artificial Intelligence for Everyone from Deeplearning.AI, Influenza Prevention and Control from World Health Organization. Attended workshops related to Bioinformatics at the University of Kerala. 3 years of experience in website management. Experience in WordPress, Blogger, Google Analytics, and Google Search Console.
