Thin-layer chromatography (TLC)

Principle Thin layer chromatography:

TLC (Thin-layer chromatography) is a chromatography technique for separating dissolved non-volatile chemical substances by the benefit of their differential migration over glass plates or plastic sheets coated with a thin layer of a excellently ground adsorbent, such as silica gel or alumina.

TLC is based on the classic chromatography principle, in which mixture components are separated by differential affinities between a fixed stationary phase and a liquid mobile phase.

Process of Thin-layer chromatography (TLC)

  • A sample of the mixture to be separated is deposited near one end of the plate for thin-layer chromatography, and a suitable solvent is allowed to rise up the plate by capillary action.
  • Because to their different degrees of attachment to the coating material on the plate or sheet, the components of the sample get separated from one another.
  • The solvent is then allowed to evaporate, and the position of the separated components is determined, usually by using reagents that react with the substances to generate coloured compounds.
  • Thin-layer chromatography has a diverse benefit over paper chromatography in that the TLC  plate or sheet is able to withstand color-forming agents & strong solvents.
Thin-layer chromatography -TLC

How does thin layer chromatography work?

  • The stationary phase – silica gel
  • Silica gel is a made up of silicon dioxide (silica). The silicon atoms are linked via oxygen (O-) atoms in a massive covalent structure. However, at the surface of the silica gel, the Si (silicon) atoms are attached to -OH (Alcohol) groups.
  • So, at the surface of the silica gel get Si-O-H bonds instead of Si-O-Si bonds. The figure shows a small portion of the silica surface.
  • Due to the -OH (Alcohol) groups, the silica gel’s surface is extremely polar and capable of forming hydrogen bonds with appropriate molecules nearby as well as van der Waals dispersion forces and dipole-dipole attractions.
  • Alumina, or aluminium oxide, is the other widely utilised stationary phase. The surface of this also has -OH (Alcohol) groups connected to the aluminium atoms. Therefore, what we say about silica gel also applies to alumina.

Retention Factor (Rf) in Thin layer chromatography

  • The retention factor (Rf) is used to record compound movement across the TLC plate.
  • Rf is calculated by dividing the overall distance travelled by the solvent by the distance travelled by each individual component. Its value is always in the range of zero to one.

Rf  = Distance travelled by component/ Distance travelled by solvent

For example, if the X component travelled 2.0 cm from the baseline while the solvent had traveled 6.0 cm, then the Rf value for the X is:

Rf = 2/6= 0.33

  • The slower a chemical travels up the TLC plate, the stronger its binding to the stationary phase adsorbent.
  • Because TLC adsorbents are often polar, non-polar molecules move faster up the plate and have larger Rf values, whereas polar compounds move slower and have lower Rf values.

Advantages of Thin Layer Chromatography (TLC)

  • It is a cheaper chromatographic technique.
  • It is an easy method with a quick growth period, and it makes it simple to see isolated compound spots.
  • It aids in the isolation of the majority of compounds.
  • The separation process moves more quickly, and the chemicals are more selective (even small differences in chemistry are enough for clear separation).
  • It is simple to evaluate the purity standards of the provided sample.
  • It is a less expensive chromatographic method.

Limitations of Thin Layer Chromatography (TLC)

  • It is difficult to differentiate between enantiomers and some isomers in TLC.
  • The Rf values of the compounds of interest must be known in advance in order to identify individual compounds.
  • Long stationary phases are not present in TLC plates. As a result, the separation time is shorter than with other chromatographic methods.

What if the substances of interested in are colourless in Thin Layer Chromatography?

Two procedures can be applied to this situation.

  1. Using fluorescence
  2. Showing the spots up chemically

Using fluorescence:

  • The stationary phase on a thin layer plate often has an element added to the plate which will fluoresce when put into UV light. As a result of UV light on it, the plate will glow.
  • Even if the spots on the final chromatogram are undetectable to the eye, that glow is covered at the location where they exist. As a result, everything on the plate will glow except for the spots when exposed to UV light.
  • The spots are demonstrated as darker spot patches.
  • While the UV is “ON” on the plate, the user will mark the positions of the spots by drawing a pencil circle around them manually. Switching the “OFF” UV source, the spots will disappear.
  • UV Cabinet will be used for this process.
Thin layer chromatography-Detection of Colourless Substance

Showing the spots up chemically

  • In some circumstances, it might be feasible to make the spots visible by causing them to react with something that yields a coloured result. Chromatograms made from combinations of amino acids provide a nice illustration of this.
  • After allowing the chromatogram to dry, a ninhydrin solution is sprayed upon it. Ninhydrin produces coloured molecules, primarily brown or purple, when it interacts with amino acids.
  • In a different procedure, the chromatogram is once more allowed to dry before being put inside a closed container containing a few iodine crystals. A container like this could be a beaker covered in a watch glass.
  • The chromatogram’s spots may interact with the iodine vapour in the container, or it may just stick to the spots more than the rest of the plate. In either case, the compounds that interest you can appear as brown patches.

Applications of TLC:

  • Pharmaceuticals, clinical diagnostics, environmental toxicity, food, water, and pesticide analyses, and cosmetics are just a few of the sectors and scientific fields that use TLC.

Distinctive applications of TLC include:

  • Food and environmental samples were tested for drug residues and antibiotics.
  • Colors, components, preservatives, and sweetening agents in food and cosmetic items are identified and quantified.
  • Pharmaceutical formulations are subjected to quality control and purity testing.
  • Prior to HPLC, rapid, high-throughput screening is performed.
  • Checking for the completion of chemical reactions

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