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What is thin layer chromatography?
A scientific method used to differentiate components in stable compounds is termed Thin Layer Chromatography. Chromatography is carried out on plastic, aluminium foil, or glass covered by a fine layer of absorbent material.
The material employed mainly by scientists here is silica gel, cellulose, or aluminium oxide. This test is a swift and straightforward process for examining an admixture of different organic solutions.
In the analysis process, a small drop of the compound in question is positioned near the bottom of the dish (this is called the stationary phase) on top of the silica gel. Gradually, the organic solvents go up the plate (this is the mobile phase).
Based on their polarities, some components have a greater affinity for silica gel, which is very polar, and other components gravitate towards the solvent readily. Thus the parts of the mixture will move from or into the plate at different speeds.
The mixture’s components will partition between the mobile and the stationary phase as the chromatography progresses. Once the chromatography is complete, components in the mixture appear as stains differentiated vertically. Each “stain” has a retention factor (Rf) which is written as:
Retention Factor = distance covered by the sample/distance covered by the solvent
The aspects affecting the retention factor are temperature, the choice of the solvent, the absorbent, and the amount of material (stain) spotted.
Thin Layer Chromatography is one of the swiftest, simplest, and inexpensive chromatography procedures. Like other chromatography processes, TLC is also based on the principle of separation.
This principle is dependent on the relative affinity of the different components of the mixture towards the two phases. The components in the mobile phase move towards the stationary phase.
This movement occurs so that the components that have more affinity towards the stationary phase travel slowly but the other components move faster. Therefore, a partition in the compound is achieved.
Once the separation process is complete, the individual elements in the compound appear as stains at different levels on the dish. Certain detection procedures can identify their nature and properties.
How to Conduct the Test?
Before conducting the test, one must know about the stages involved and the prerequisite required to carry out the process. First, one needs to have very thin dishes or plates for the chromatography.
For this, ready-made plates must be picked that are chemically stable and non-volatile. To the surface of this stationary phase, a thin coating of silica gel is applied in the form of absorbent material.
Next, the Thin Layer Chromatography Chamber is utilized to develop dishes responsible for maintaining a safe environment inside which the “stains” from the experiment will continue developing. It also prevents evaporation of the solvent and keeps the experiment area clean and non-hazardous.
Next, one must pay attention to the chromatography mobile phase, which contains and manages the contents of a solvent or a mixture of solvents. This phase should be free of pollutants. The greater the purity, the better the stains that come out developed.
Another important accessory for this phase is the filtration paper. Thin Layer Chromatography filtering papers must be present for the test. The paper must be dampened with water before being placed in the mobile phase.
In contrast to this, the stationary phase placed on the plate must be dried out after being sterilized. Thin lines are drawn at the bottom of the dish with a marker or a pencil. This is where the participants need to put the sample stains.
Place the mobile phase inside the experiment chamber and put a dampened filtration paper on the mobile phase to control the humidity. Then the dish is placed in the experiment chamber and shut properly with a lid. It is kept in such a way that the sample faces the mobile phase.
Finally, the participants can now put the dish for the development of stains and wait. Once the stains are visible clearly and take the dishes out to dry. The stains developed can be studied using a UV light chamber.
Its Uses and Applications
Thin Layer Chromatography has numerous applications such as:
- The thorough testing of numerous medicines like antihistamines, anticonvulsant tranquillizers, sedatives, local anaesthesia, hypnotics, analgesics, and steroids.
- It is widely used in Biochemical experiments like the isolations or separation of biochemical particles from body fluids such as urine, sperm, and blood plasma.
- Thin-layer chromatography is also utilized to recognize natural products such as glycosides, alkaloids, waxes, fixed oil, volatile oil, or essential oils.
- It finds use in the food industry to identify preservatives, sweetening agents, and food colouring.
Author Bio: Nora George is a freelance writer and extremely fond of anything related to Digital Marketing and Business. She is writing Technologies as well as fiction, like good music, loves her cat and eats too much. More than anything, She loves to share the knowledge of Technology.
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