Thin layer chromatography – Comprehensive Guide
This article describes about working principle of Thin Layer Chromatography, types of Mobile and stationary phases, Sample application techniques, Visualization of plates, Applications, Advantages and limitations. Enlisted List of frequently asked questions.
What is mean by Thin Layer Chromatography and its Working principle?
Thin layer chromatography (TLC) is a simple and efficient analytical technique used to separate and identify the components of a mixture .
Its principle is based on the differential migration of components of a sample mixture on a stationary phase in the presence of a mobile phase. The stationary phase, which is coated onto a plate, is typically a thin layer of an adsorbent material, such as silica gel, alumina, or cellulose.
The sample is applied to the stationary phase, and the plate is then placed in a container with a solvent system. As the solvent moves up the plate by capillary action, it carries the components of the sample with it.
Each component interacts differently with the stationary and mobile phases, causing them to migrate at different rates. This differential migration results in the separation of the components of the sample mixture. TLC also has its limitations, and its results should always be interpreted with caution
Types of Stationary Phases used in Thin Layer Chromatography (TLC) ?
The choice of the stationary phase depends on nature of the sample and the separation requirements. The most commonly used stationary phases in TLC are silica gel, alumina, and cellulose.
Silica Gel
Silica gel is the most widely used stationary phase in TLC. It is available in different particle sizes, shapes, and pore sizes, making it suitable for a wide range of applications. Silica gel has a high surface area and a high affinity for polar compounds.
Alumina
Alumina is another commonly used stationary phase in TLC. It has a lower surface area than silica gel but a higher affinity for non-polar compounds.
Cellulose
Cellulose is a stationary phase that is high surface area and a high affinity for polar compounds.
Types of Mobile Phases Used in Thin Layer Chromatography (TLC) ?
The choice of the mobile phase depends on the polarity of the sample and stationary phase. The most commonly used mobile phases in TLC are non-polar solvents, polar solvents, and two-phase solvent systems.
Non-polar Solvents
Non-polar solvents, such as hexane, benzene, and toluene, are used for the separation of non-polar compounds on a polar stationary phase.
Polar Solvents
Polar solvents, such as ethanol, methanol, and water, are used for the separation of polar compounds on a non-polar stationary phase.
Two-Phase Solvent Systems
Two-phase solvent systems are used for the separation of compounds with intermediate polarities. Examples of two-phase solvent systems are chloroform-methanol, ethyl acetate-methanol-water, and butanol-acetic acid-water.
Techniques used for Sample Application in Thin Layer Chromatography (TLC)
The choice of the technique for sample application depends on nature of the sample and the desired resolution. The most commonly used techniques for sample application are spotting, spraying, and dipping.
Spotting
Spotting is the simplest and most commonly used technique for sample application. A small amount of the sample is placed on the plate with a capillary tube or a micro syringe.
Spraying
Spraying is a technique that is used for the application of samples that are difficult to dissolve or are present in low concentrations. The sample is sprayed onto the plate using a fine mist sprayer.
Dipping
Dipping is a technique that is used for the application of samples that are in a liquid form. The plate is dipped into the sample solution, and the solvent is then evaporated.
Visualization of Thin Layer Chromatography (TLC) Plates
The visualization of TLC plates is important for the detection and identification of separated components. The most commonly used visualization techniques are UV absorbance, staining, and charring.
UV Absorbance
UV absorbance is a widely used technique for the visualization of TLC plates. The separated components on the plate can be detected by exposing the plate to UV light. Compounds that absorb UV light will appear as dark spots on a light background.
Staining
Staining is a technique that is used for the detection of compounds that do not absorb UV light. A staining reagent is sprayed onto the plate, and the plate is then heated or left to dry. Compounds that react with the staining reagent will appear as colored spots on a light background.
Charring
Charring is a technique that is used for the detection of compounds that are not visible with UV absorbance or staining. The plate is heated until the compounds on the plate char. The charred compounds appear as dark spots on a light background.
Applications of Thin Layer Chromatography (TLC)
TLC is used in the quality control of pharmaceuticals for the identification of active ingredients, impurities, and degradation products.
it can be used for semi-quantitative analysis by comparing the Rf values of the sample components to those of known standards. However, it is generally less accurate and precise than other chromatographic Techniques, such as HPLC.
Advantages of Thin Layer Chromatography (TLC)
TLC has many advantages over other separation techniques, Majorly some of them enlisted as below :
- It is a simple and low-cost technique.
- It requires small amounts of sample and solvent.
- It is a rapid technique, with separations typically completed in minutes.
- It can separate a wide range of compounds with different polarities.
Limitations of Thin Layer Chromatography (TLC)
TLC also has some limitations, includes,
- It is a qualitative technique and does not provide accurate quantitative information.
- The separation resolution may not be as high as with other techniques, such as high-performance liquid chromatography (HPLC).
- The separation efficiency may decrease with the use of a large number of samples on a single plate.
Frequently Asked Questions (FAQs)
What is the principle of thin layer chromatography?
The principle of thin layer chromatography is based on the differential migration of components of a sample mixture on a stationary phase in the presence of a mobile phase.
What are the types of stationary phases used in Thin Layer Chromatography (TLC) ?
The most commonly used stationary phases in TLC are silica gel, alumina, and cellulose.
What are the types of mobile phases used in TLC?
The most commonly used mobile phases in TLC are non-polar solvents, polar solvents, and two-phase solvent systems.
What are the advantages of Thin Layer Chromatography (TLC) ?
The advantages of thin layer chromatography include simplicity, low cost, small sample and solvent requirements, rapid separations, and the ability to separate a wide range of compounds with different polarities.
Can Thin Layer Chromatography (TLC) is used for quantitative analysis?
While TLC is primarily a qualitative technique, it can be used for semi-quantitative analysis by comparing the Rf values of the sample components to those of known standards. However, it is generally less accurate and precise than other chromatographic techniques, such as HPLC.
What is the difference between Thin Layer Chromatography (TLC) and Paper Chromatography?
TLC and paper chromatography are both chromatographic techniques used for separating and analyzing components of a mixture. However, TLC uses a thin layer of stationary phase material coated on a plate, while paper chromatography uses a strip of filter paper as the stationary phase. TLC is generally more accurate and precise than paper chromatography.
What is the difference between Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) ?
TLC and HPLC are both chromatographic techniques used for separating and analyzing components of a mixture. However, HPLC uses a packed column as the stationary phase and a high-pressure liquid as the mobile phase, while TLC uses a thin layer of stationary phase material coated on a plate and a liquid or gas as the mobile phase. HPLC is generally more accurate and precise than TLC, but also more expensive and time-consuming.
