Thin Layer Chromatography(TLC) is a separation technique used to separate the components of non-volatile mixtures. TLC is significantly a better separation process than Paper chromatography.
TLC was first reported to be used in 1938 in Izmailov and Schreiber.
Principle of Thin Layer Chromatography
Thin Layer Chromatography is a separation process based on the principle of adsorption in which the components of sample separate based on their affinity towards the stationary and mobile phase where the stationary phase is a thin layer of adsorbent material such as Aluminium oxide, Cellulose, or Silica gel while the mobile phase is the solvent.
The separation occurs in a way that the components of higher affinity in a mixture move faster than those which have less affinity towards the stationary phase. When the solvent reaches the solvent front the components are placed at different Rf values and thus the separation is achieved.
This procedure of TLC is similar to Paper Chromatography. The only difference is the stationary phase.
Method/Procedure of Thin Layer Chromatography
The procedure of Thin Layer Chromatography is as follows:
1. Preparation of TLC plate
The ready-made TLC plates are available commercially. The ready-made plates are made of a thin layer of silica gel on Aluminium plates.
The TLC plates can also be made in a lab. To prepare a TLC plate Silica gel is mixed with Gypsum and water to make a thick slurry. Then apply the slurry on a clean glass plate uniformly. Uneven thickness of slurry on the plate can alter the results. After evenly placing the slurry on the place air-dry it.
2. Activation of TLC plate
Take a TLC plate and activate it by keeping it in a hot oven for about 20-30 minutes at 110 degree celsius.
3. TLC Chamber
TLC chamber is used to develop the chromatogram. Before proceeding to the development, keep the solid in the TLC chamber for about half an hour for saturation of molecules of solvent.
4. Spotting the sample
Draw a baseline above 4 cm from one end of a TLC plate. Spot the sample on that baseline at a fair distance such that during the development of the sample, the components of one sample don’t mix with the other.
5. Development of Chromatogram
Place the TLC plate in the chamber such that the baseline doesn’t come in contact with the solvent. The solvent (mobile phase) will run in the upward direction through capillary action and in the process, the components of the sample will move upwards according to their affinity towards the stationary and mobile phase.
6. Visualisation of Chromatogram
After the solvent has reached the solvent front, the plate is taken out and air dried. Then the developed chromatogram is visualized using UV light or visualizing agents such as KMnO4 stain, Dragendorff’s reagent, etc.
Applications of Thin Layer Chromatography
- Testing of medicines such as sedatives, hypnotics, Tranquilizers, etc.
- Separation of amino acids, organic compounds, and inorganic ions.
- To check the purity of the sample.
- To check the presence of preservatives in a product.
- For analysis of biochemical substances such as separation of biochemical metabolites from Urine, Blood Plasma, and bodily fluids, etc.
- To identify natural products such as oils, alkaloids, and waxes, etc.
- To track the progress of a reaction.
- Separation of inks, colors, preservatives, sweetening agents, etc.
Advantages of Thin Layer Chromatography
- Highly-sensitive with accurate results.
- Inexpensive technique.
- Requires a very small amount of sample even possible in microlitre.
- Fast and can separate multiple samples simultaneously.
- Components can be visualized with the help of UV light.
- Visualizing agents such as corrosive reagents can help in better detection of components.
- Quantitative analysis is also possible with the help of the standard curve.
- Can help in the identification of compounds.
- TLC makes it easy to analyze the purity standards of a sample.
- The separated components can also be extracted by simply scraping the powder from that area of the plate.
- TLC can perform even in high temperatures, thus giving it an extra advantage over other techniques that don’t give accurate results in high temperatures.
Disadvantages of Thin Layer Chromatography
- The length of the stationary phase is smaller thus providing a shorter length of separation of samples.
- Results produced are difficult to reproduce.
- Quantitative analysis is quite difficult.
- Since, it is an open system, the humidity, the temperature can have a significant effect on the outcome.
- The detection limit is too high. Therefore, using TLC for a low detection limit is not possible.
- Separation of only soluble components of mixtures is possible.
- TLC cannot determine the difference between isomeric and enantiomeric forms of the compound.
- The requirement of knowing the Rf value of a compound before analysis is another disadvantage. This is because an unknown compound can be detected only if we know the Rf value of that compound beforehand so that it can be matched.
- While preparing the TLC plate, if the thickness of the slurry is not uniform, the outcome may not be the same.
- It is limited to only non-volatile compounds.
- TLC is not automatic.