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Basic Mechanism of Thin Layer Chromatography


Thin Layer Chromatography:

 Defination of Thin Layer Chromatograpy


Thin layer chromatography (TLC) is a technique  which is used for analysis of mixtures by separating into compounds  which are present within the mixture.
Applications of thin-layer chromatography:
TLC is usefull in the determination of large number of components along  a mixture  For the identification and purification of a compound.
 Nature of Thin Layer Chromatography:
TLC is a receptive method  measures the sample even in  microgram (0.000001 g) quantities can be analyze by TLC  and it also takes minute time for an analysis  just for 5-10 minutes.
Mechanism of Thin Layer Chromatography:

TLC has following mechanism:
 Mechanism of thin-layer consists of following three steps named as following
Spotting development and visualization.


Preparation of sample solution:
First step is that the sample which is  to be analyzed is soften in a volatile ( which can be easily evaporated) solvent  for the production of a very dilute solution which is about to 1%.

Spotting Process:
Spotting procedure includes the use of a micro pipet for the passage of little amount of already prepared dilute solution at one corner of a TLC plate in this condition a thin layer of silica gel   in powdered form  which has been caked onto the sheet which is made up of plastic. The spotting solvent evaporates at a greater speed and makes a small spot of given material behind itself.     
Process of development:
Process of development consists of putting at the foundation of the TLC plate into a superficial tank of a development solvent,  after this it travels to the top by the process of capillary action. As the solvent moves to the top of the plate it travels over the original spot. A war is set up between the plate of silica gel and the development solvent for the spotted material.
Action of silica gel:

The silica gel which is more polar try to grasp the spot to its original place and the solvent tries to pull the spot along with it as it moves towards the top of the plate. The result depends on a sense of balance among  following three polarities  one of them is that of the plate second is the development solvent and the third is spotted material.

Action of polarity:

If the development solvent is more polar the spot will travel at  some distance from its original position. Different components of the original spot, varying in their polarities, will travasl to some distances from the original spot position and give a result in the form of separate spots.
Marking of the solvent front:
When the solvent has  moved almost to the apex of the plate after this the plate is removed, the solvent front distinct with a pencil, and the solvent is permitted to evaporate.
Process of visualization :

For colored compounds:
Visualization process for those compound which are  present within  colored compounds have  easy and simple method. These spots can be frankly seen after developmental process.
For colorless compounds:

However most compounds are colorless so for them  visualization process is wanted. The silica gel  which is present on the TLC plate is saturated with a fluorescent material that glitter under ultraviolet (UV) light. A spot will get in the way with the fluorescence and observed as a dark spot on a lustrous background.

Marking of solvent front under UV  light:
Under the UV light, the spots can be marked with a pencil to stain their position.
Second method of visulization:


Another technique for the process of visualization is skilled by keeping a  plate into  vapors of iodine for some time.
Color of the organic compounds:
 A large number of organic compounds will make a dark-colored complex with iodine.
 It is better to use as a minimum two visualization methods if in case of such compound which  does not show up with one particular method.
Rf value:
The Rf value is used to tell the transferring of the materials within the plate. Rf is equivalent to the distance travel by a substance overby the distance travel by the solvent.
Rf value in numbers:
Its value is always between zero and one. Comparing these Rf's with the Rf's of known compounds might enable a tentative identification to be made.

Observation of spots:
Observation of three spots means there are  only three components  present in the mixture. Some components have same polarities so they xcan be seen in the form of one spot after the process of development.

Development solvent with high polarity:
If a development solvent having high polarity is used, all the  components in the mixture will travel within the solvent and no separation will  take place Rf value will also be greater.
Development solvent with low polarity:


If the solvent has low polarity then components will not travel as much as necessary  and separation process cannot be repeated also Rf value will be so small. Usually an efficient solvent is one that gives Rf value within the choice of 0.3 - 0.7.

Note that the spotting of  solvent is used in a simple process as a medium to shift the material for the analysis on the TLC plate. Once the reassign is made after this spotting solvent evaporate.



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