Chromatography is a vital biophysical method that allows it to identify, separate and purification of the constituents of a mix for an analysis that is both qualitative as well as quantitative. For this type of physical separation, the components that are to be separated are divided into two phases. One has a stationary (stationary phase) while the second (the mobile phase) is moving in a specific direction. Based on what phase is stationary and which the mobile phase, they may be of various types. Thin Layer Chromatography is described as a technique for delineating or separating an assortment of components to separate them into components, making use of adsorbents with finely separated adsorption or (liquid) placed on the surface of a plate, and liquid as mobile phase.
Principle of Thin Layer Chromatography (TLC)
Thin-layer chromatography takes place on a plastic, glass or aluminium foil that is then covered with a very thin layer an adsorbent, typically silica gel or aluminum oxide (alumina) or cellulose. The adsorbent layer is called the stationary phase. After the sample is placed on the plate the solvent or the solvent combination (known as mobile) is drawn through the plate through capillary action. Because different chemicals ascend through the TLC plate with different speeds, separation can be attained.
This is built on the concept of adsorption or partition chromatography, or a combination of both, depending on the adsorbent as well as the treatment it undergoes and the kind of the solvent used. Components with greater affinity for stationary phase move slower. Components that have less affinity to stationary phase move more quickly. After separation when the component is separated, they appear as spots at an appropriate distance in the plates. Their character or nature is recognized by the appropriate detection methods.
Components of Thin Layer Chromatography (TLC)
TLC system components consists of:
- TLC plates, preferably ready made with a stationary phase: They are stable and chemically inert, which have a thin layer of stationary phase is applied to the entire surface. The stationary phase of the plates is uniform in thickness and has small particles.
- TLC chamber– This is used to create TLC plate. The chamber provides a consistent interior for the an effective formation of spots. It also helps prevent the evaporation of solvents and helps keep the process clean.
- Mobile phase– This constitutes a solvent or solvent mixture. The mobile phase that is utilized should not contain any particulates and be with the highest purity to ensure the proper creation of TLC spots. The solvents that are recommended are chemically inert and compatible with the sample, and a stationary phase.
- A filter paper- It is moistened during the mobile phase to be put inside the chamber. This creates a uniform rise in the mobile phase across all the distance of the stationary phase.
Procedure of Thin Layer Chromatography (TLC)
It is then sprayed on the plate evenly and is allowed to dry and become stable. Today, however ready-made plates are increasingly employed.
- Utilizing an eraser, a fine trace is created on the lower edge of the plate in order to allow the spot to be applied.
- Then, solutions samples are applied to the spots drawn on the line at equally spaced distances.
- It is then poured through the TLC chamber, which is then leveled a just a few centimeters over the chamber’s at the bottom.
- A dampened filter paper in mobile phase is positioned on the inside of the chamber in order to ensure an equal amount of humidity (and can also prevent edges effect).
- The plate that has been prepared with sample spotting is put into the TLC chamber in such a way that the face of the plate with the sample line faces towards the cell phase. The chamber is then closed by using the lid.
- After that, the plate gets submerged in the solvent, so that the sample spots are above the mobile phase (but not completely submerged in solvent) to allow for the development.
- The proper time frame is provided for the formation of spots.
- The plates are removed and dried.
- The spots of the sample are examined in a suitable ultraviolet light chamber or other method that is recommended for the sample.
A few common methods for visualizing the outcomes of TLC plates include: TLC plate comprise
- UV light
- Iodine Staining is extremely efficient in detecting carbohydrates because it is black when in contact with Iodine
- KMnO4 stain (organic molecules)
- Ninhydrin Reagent is often used to determine amino acids and proteins.
Retention Factor (Rf ) Value
- The behavior of a compound on an TLC is typically described using its relative mobility, or Rf value.
- Rf , also known as Retention factor, is a distinct amount for all compounds that is under similar conditions.
- The Rf for an ingredient is steady from experiment one to following in the case that the chromatography conditions are also constant.
- Solvent system
- the thickness of the of the
- Quantity of material found
- Because these variables are hard to maintain constant between experiments, the relative Rf values are typically regarded.
- Relative Rf” refers to the fact that the reported values are compared to an established standard.
- Rf value is determined by using Rf Value is computed by using an equation like.
Applications of Thin Layer Chromatography (TLC)
- Monitoring the development of reactions
- Find the components that are present in a particular mixture
- Verify the pureness of a material.
- Analyzing the ceramides as well as the fatty acids
- Identification of insecticides or pesticides in food or water
- Analyzing the composition of dyes in fibers used in the field of forensics
- Verifying the purity of radiochemicals of radiopharmaceuticals
- Identification of herbal medicinal plants as well as their components
Advantages of Thin Layer Chromatography (TLC)
- It’s a simple procedure with a brief development time.
- It assists in the visualisation of compounds that have been separated.
- It assists in isolating the majority of compounds.
- The process of separation is quicker and the selection of compounds is better (even minor differences in the chemical chemistry are enough to allow for the clearest separation).
- The standards for purity of the sample are easily assessed.
- It’s a less expensive method of chromatography.
Limitations of Thin Layer Chromatography (TLC)
- It is difficult to distinguish between isomers and enantiomers.
- To determine the exact compounds to identify specific compounds, they must be identified by their Rf numbers for each compound of interest should be identified prior to the time of identification.
- TLC plates don’t have lengthy stationary phases. This means that the separation length is not as long as other techniques for chromatography.