Thin Layer Chromatography- Definition, Principle, Parts, Steps, Uses

Chromatography is a technique that is used to separate and analyze the components of a mixture based on their different physical and chemical properties. Chromatography can be used for various purposes, such as:

• Identifying the components of a mixture
• Determining the purity of a substance
• Isolating and purifying a desired compound
• Separating and quantifying the components of a mixture
• Studying the interactions between molecules and different phases

The basic principle of chromatography is that a mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various components of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and the stationary phases. Different types of chromatography use different stationary and mobile phases and different modes of separation.

Some common types of chromatography are:

• Paper chromatography: The stationary phase is a paper sheet and the mobile phase is a solvent.
• Column chromatography: The stationary phase is a solid adsorbent packed in a column and the mobile phase is a solvent.
• Gas chromatography: The stationary phase is a thin layer of liquid or polymer on an inert solid support inside a column and the mobile phase is an inert gas.
• Liquid chromatography: The stationary phase is a solid or liquid on an inert solid support inside a column and the mobile phase is a liquid.
• Thin layer chromatography: The stationary phase is a thin layer of solid adsorbent coated on a glass, plastic or metal plate and the mobile phase is a solvent.

In this article, we will focus on thin layer chromatography (TLC), which is one of the simplest and most widely used chromatographic techniques in organic chemistry. TLC can be used to separate and identify compounds, monitor reactions, determine purity, and more. We will discuss the definition, principle, components, procedure, retention factor, applications, advantages and limitations of TLC in detail.

Thin Layer Chromatography (TLC) is a type of chromatography that uses a thin layer of solid material as the stationary phase and a liquid as the mobile phase. TLC is a simple, fast and inexpensive technique that can separate and identify the components of a mixture based on their different affinities to the stationary and mobile phases.

TLC can be performed on a sheet of glass, plastic or metal that is coated with a thin layer of adsorbent material, such as silica gel, alumina or cellulose. The adsorbent material acts as the stationary phase that retains some components of the mixture more than others. The sheet is called the TLC plate.

The mixture to be separated is applied as small spots near the bottom edge of the TLC plate. The plate is then placed in a closed container that contains a small amount of solvent or solvent mixture. The solvent acts as the mobile phase that moves up the plate by capillary action. As the solvent rises, it carries along the components of the mixture at different rates depending on their solubility in the solvent and their adsorption to the stationary phase.

The separation of the components is achieved when they reach different heights on the plate. The components that are more soluble in the solvent and less adsorbed to the stationary phase will move faster and farther than those that are less soluble and more adsorbed. The components will appear as distinct spots on the plate at different positions.

The spots can be visualized by various methods, such as UV light, iodine vapour or chemical reagents. The identity and purity of the components can be determined by comparing their positions and colours with those of known standards or reference materials. The relative mobility of each component can be expressed by its retention factor (Rf) value, which is calculated by dividing the distance travelled by the component by the distance travelled by the solvent front.

TLC is widely used for qualitative analysis of organic and inorganic compounds, such as drugs, pesticides, dyes, lipids, amino acids and carbohydrates. TLC can also be used for quantitative analysis by measuring the intensity or area of the spots. TLC can provide information about the number, nature and purity of the components in a mixture. TLC can also be used to monitor the progress of chemical reactions or to isolate pure compounds from a mixture.

Thin layer chromatography (TLC) is based on the principle of differential migration of the components of a mixture on a stationary phase under the influence of a mobile phase. The stationary phase is a thin layer of an adsorbent material, such as silica gel, alumina, or cellulose, that is coated on a flat support, such as glass, plastic, or aluminium foil. The mobile phase is a solvent or a solvent mixture that moves along the stationary phase by capillary action.

The separation of the components depends on their relative affinity towards the stationary and mobile phases. The components that have a higher affinity for the stationary phase will move slower and remain closer to the origin (the point where the sample is applied). The components that have a higher affinity for the mobile phase will move faster and travel farther from the origin. The degree of separation also depends on the polarity, solubility, and molecular size of the components.

The separation process can be monitored by visualizing the spots of the components on the stationary phase. This can be done by using a UV light chamber, if the components are fluorescent or have been treated with a fluorescent dye, or by using various staining or spraying techniques, if the components are not fluorescent. The identity and purity of the components can be determined by comparing their retention factor (Rf) values with those of known standards. The Rf value is the ratio of the distance travelled by a component to the distance travelled by the solvent front.

TLC can be classified into two types based on the principle involved: adsorption TLC and partition TLC. Adsorption TLC is when the separation is mainly due to the difference in adsorption of the components on the stationary phase. Partition TLC is when the separation is mainly due to the difference in partitioning of the components between the stationary and mobile phases. In practice, most TLC systems involve a combination of both adsorption and partition mechanisms.

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 a fine particle size. The most common stationary phases are silica gel, aluminium oxide (alumina), or cellulose. These materials have different polarities and affinities for the analytes, which affect the separation efficiency.

• TLC chamber: This is used for the development of TLC plate. The chamber maintains a uniform environment inside for proper development of spots. It also prevents the evaporation of solvents, and keeps the process dust free. The chamber can be made of glass, plastic, or metal, and should have a tight-fitting lid.

• Mobile phase: This comprises of a solvent or solvent mixture that moves up the plate by capillary action. 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, and the TLC plate. The choice of the mobile phase depends on the polarity and solubility of the analytes. A more polar solvent will move faster and farther up the plate, while a less polar solvent will move slower and shorter.

• 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. It also helps to saturate the chamber with solvent vapors, which reduces evaporation and improves reproducibility.

• A pencil: This is used to mark a thin line at the bottom of the plate to apply the sample spots. The pencil marks should be light and thin, as they may interfere with the detection of some analytes.

• A capillary tube: This is used to apply small amounts of sample solutions on the spots marked on the line in equal distances. The capillary tube should be clean and dry, and should not touch the surface of the plate. The sample spots should be small and concentrated, as they will spread during development.

• A UV lamp or other detection methods: This is used to visualize the separated spots on the plate after development. Some analytes may fluoresce under UV light, while others may need to be stained with specific reagents to become visible. The detection methods depend on the nature and properties of the analytes.

The procedure of thin layer chromatography (TLC) involves the following steps:

• Preparation of TLC plate: The TLC plate is a sheet of glass, plastic, or aluminium foil that is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. The adsorbent layer should be uniform and smooth, and its thickness should be about 0.1 to 0.25 mm. The TLC plate can be prepared by spreading the adsorbent slurry on the plate and drying it in an oven, or by using commercially available ready-made plates. A pencil line is drawn about 1 cm from the bottom edge of the plate to mark the origin, where the sample spots will be applied.

• Application of sample spots: The sample solutions are applied on the origin line using a capillary tube, a microsyringe, or a spotting device. The spots should be small and well-defined, and spaced at least 1 cm apart to avoid overlapping. The amount of sample applied should be proportional to its concentration and polarity. The more polar and concentrated the sample is, the less amount should be applied. The TLC plate is dried after spotting to evaporate the solvent.

• Development of TLC plate: The TLC plate is placed in a developing chamber that contains the mobile phase, which is a solvent or a mixture of solvents. The mobile phase should be chosen based on its polarity and compatibility with the sample and the stationary phase. The more polar the mobile phase is, the faster the sample components will move up the plate. The developing chamber should be saturated with the mobile phase vapor before inserting the TLC plate, to ensure a uniform atmosphere and prevent evaporation. A filter paper soaked in the mobile phase can be placed on the inner wall of the chamber to help with saturation. The TLC plate is positioned vertically in the chamber, with the origin line slightly above the level of the mobile phase. The chamber is covered with a lid to avoid contamination and disturbance. The mobile phase rises up the plate by capillary action and carries along the sample components at different rates, depending on their polarity and affinity to the stationary phase. The more polar a component is, the more it will stick to the stationary phase and move slower. The less polar a component is, the more it will dissolve in the mobile phase and move faster. The development is stopped when the solvent front reaches about 1 cm from the top edge of the plate.

• Visualization of TLC spots: The TLC plate is removed from the chamber and dried in air. The spots of the separated components are visualized using different methods, depending on their nature and properties. Some components may be visible to the naked eye or under UV light, while others may require chemical staining or spraying with specific reagents. For example, iodine can be used to detect carbohydrates, ninhydrin can be used to detect amino acids and proteins, and KMnO4 can be used to detect organic molecules. The spots can be marked with a pencil for further analysis.

• Calculation of Rf values: The Rf value (retention factor) is a measure of how far a component has moved relative to the solvent front on the TLC plate. It is calculated by dividing the distance traveled by the component from the origin (d) by the distance traveled by the solvent front from the origin (D). Rf = d/D. The Rf value is a characteristic of each component under specific experimental conditions, such as solvent system, adsorbent type, temperature, etc. It can be used to identify unknown components by comparing them with known standards or reference values.

The behaviour of a compound on a TLC plate is usually described in terms of its relative mobility or Rf value. Rf stands for retention factor and it is a unique value for each compound under the same conditions. The Rf value indicates how far a compound travels on the TLC plate relative to the solvent front. The higher the Rf value, the more soluble the compound is in the solvent and the less it is adsorbed to the stationary phase. The lower the Rf value, the more it is adsorbed to the stationary phase and the less it is soluble in the solvent.

The Rf value is calculated using the following equation:

$$R_f = \frac{d_s}{d_f}$$

where $d_s$ is the distance travelled by the compound spot from the origin and $d_f$ is the distance travelled by the solvent front from the origin.

The Rf value is a constant from one experiment to another only if the chromatography conditions are also constant. These conditions include:

• solvent system
• adsorbent
• thickness of the adsorbent
• amount of material spotted
• temperature

Since these factors are difficult to keep constant from experiment to experiment, relative Rf values are generally considered. Relative Rf means that the values are reported relative to a standard compound that has a known Rf value under the same conditions.

The Rf value can be used to identify compounds present in a mixture by comparing them with known standards or reference tables. It can also be used to determine the purity of a substance by checking if there is only one spot on the TLC plate or if there are any impurities present. However, some limitations of using Rf values are:

• It cannot tell the difference between enantiomers and some isomers that have similar polarity and solubility.
• It requires prior knowledge of the Rf values for the compounds of interest or access to reference tables.
• It depends on several factors that can vary from experiment to experiment and affect the accuracy and reproducibility of the results.

Thin layer chromatography (TLC) is a versatile and widely used technique for the separation, identification, and quantification of various compounds in a mixture. TLC has many applications in different fields of science, such as:

• Pharmaceutical analysis: TLC can be used to monitor the progress of reactions, determine the purity of drugs, identify impurities or adulterants, and quantify the active ingredients in pharmaceutical formulations .
• Herbal analysis: TLC can be used to analyze herbal extracts with minimum sample cleanup requirement, identify natural products like essential oils, fixed oils, glycosides, alkaloids, etc., and quantify the chemical constituents of herbal medicines .
• Food analysis: TLC can be used to detect pesticides, insecticides, preservatives, additives, or contaminants in food and water samples . TLC can also be used to analyze the fatty acid composition, lipid classes, and antioxidant activity of edible oils.
• Forensic analysis: TLC can be used to analyze the dye composition of fibers, ink samples, explosives, drugs of abuse, or other trace evidence in forensic investigations .
• Biochemical analysis: TLC can be used to separate and identify biomolecules like amino acids, proteins, lipids, carbohydrates, nucleic acids, hormones, etc. TLC can also be used to study enzyme activity, metabolic pathways, or biosynthetic processes .
• Environmental analysis: TLC can be used to monitor the pollution levels of air, water, or soil by detecting organic or inorganic pollutants like polycyclic aromatic hydrocarbons (PAHs), heavy metals, pesticides, etc .

These are some of the common applications of TLC in various domains of science. However, TLC can also be applied to other areas where separation and identification of complex mixtures are required. TLC is a simple, fast, sensitive, and cost-effective method that can provide valuable information about the chemical composition and properties of different substances.

Thin layer chromatography (TLC) is a widely used technique for the separation and identification of organic compounds. It has several advantages over other chromatographic methods, such as:

• Simplicity: TLC is easy to perform and requires minimal equipment and materials. The procedure involves spotting the sample on a TLC plate, developing the plate in a suitable solvent, and visualizing the spots under UV light or with a chemical reagent. The results can be obtained in a short time and with little training.
• Visualization: TLC allows the direct observation of the separated components on the plate. The number, position, shape, and color of the spots can provide useful information about the identity and purity of the sample. The spots can also be further analyzed by scraping them off the plate and using spectroscopic or chemical methods.
• Isolation: TLC can be used to isolate pure compounds from a mixture by cutting out the spots from the plate and eluting them with a solvent. This technique is called preparative TLC and can be useful for obtaining small amounts of compounds for further analysis or synthesis.
• Speed: TLC is a fast technique that can separate complex mixtures in a matter of minutes. The separation depends on the choice of the solvent and the stationary phase, which can be optimized for different types of compounds. TLC can also separate compounds that have similar boiling points or polarity, which may be difficult to separate by other methods.
• Selectivity: TLC can separate compounds that have small differences in their chemical structure or properties. The separation is based on the relative affinity of the compounds for the stationary and mobile phases, which can be influenced by factors such as polarity, hydrogen bonding, charge, and molecular size. TLC can also separate enantiomers and diastereomers by using chiral stationary phases or additives.
• Cost-effectiveness: TLC is a cheap technique that requires low amounts of sample and solvent. The TLC plates are reusable and can be prepared in the laboratory or purchased commercially. The solvents used for TLC are usually common organic solvents that are readily available and inexpensive.

These advantages make TLC a versatile and powerful technique for the analysis of organic compounds in various fields such as pharmaceuticals, food, cosmetics, forensics, environmental science, and natural products.

Despite its many advantages, thin layer chromatography also has some limitations that need to be considered. Some of the limitations are:

• TLC cannot separate compounds that have very similar polarity or molecular weight. This means that some compounds may co-migrate on the TLC plate and appear as a single spot, making it difficult to identify them individually.
• TLC cannot distinguish between enantiomers and some isomers, which are compounds that have the same molecular formula but different spatial arrangements of atoms. This can lead to false positive or negative results in some cases.
• TLC has a low sensitivity and resolution compared to other chromatographic techniques. This means that TLC may not be able to detect very small amounts of compounds or separate compounds that have very close Rf values. Moreover, TLC plates have a limited length of stationary phase, which limits the extent of separation that can be achieved.
• TLC is prone to errors and variations due to human factors and environmental conditions. For example, the quality and thickness of the stationary phase, the amount and composition of the mobile phase, the temperature and humidity of the chamber, and the method of spotting and visualizing the spots can all affect the outcome of TLC. Therefore, TLC requires careful standardization and calibration to ensure accuracy and reproducibility.
• TLC is not suitable for quantitative analysis, which is the measurement of the amount or concentration of a compound in a sample. This is because TLC does not provide a direct relationship between the intensity or size of a spot and the amount of a compound. To perform quantitative analysis using TLC, additional steps such as densitometry or spectrophotometry are needed, which can increase the cost and complexity of the analysis.

These are some of the limitations of thin layer chromatography that should be taken into account when choosing this technique for analytical purposes. However, TLC can still be a useful and convenient method for qualitative analysis, especially for screening and monitoring purposes.

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