THIN LAYER CHROMATOGRAPHY

By: K. N. Sai Sreenivas 14-MDM-02

Chromatography:

Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify and/or quantify the mixture or components.

Thin Layer Chromatography:

Thin layer chromatography (TLC) is a chromatography technique used to separate nonvolatile mixtures.

TLC is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary phase.

• After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.

• TLC consists of three steps :

I. Spotting II. Development and III. Visualization

I) Spotting:

Spotting consists of using a micro-pipette to transfer a small amount of this dilute solution to one end of a TLC plate, in this case a thin layer of powdered silica gel that has been coated onto a plastic sheet.

The spotting solvent quickly evaporates and leaves behind a small spot of the material.

II) Development:

The spotted plate is placed in a sealed development tank filled with vapor of mobile phase, its lower side immersed in solvent to a level below the applied sample spots. The solvent rises due to capillary flow in a process called development.

III) Visualization:

Some organic compounds are colored. If you are fortunate enough to be separating organic molecules that are coloured such as dyes, inks or indicators, then visualizing the separated spots is easy.

However, since most organic compounds are colorless, this first method does not always work. In most cases observing the separated spots by UV light works well.

TLC plates normally contain a fluorescent indicator which makes the TLC plate glow green under UV light of wavelength 254 nm.

Compounds that absorb UV light will quench the green fluorescence yielding dark purple or bluish spots on the plate. Simply put the plate under a UV lamp, and the compounds become visible to the naked eye.

Lightly circle the spots, so that you will have a permanent record of their location for later calculations.

Advantages:

• Simple process with short development time

• The separation process is faster and the selectivity for compounds is higher even small differences in chemistry is enough for clear separation

• The purity standards of the given sample can be assessed easily

• It is a cheaper chromatographic technique

Applications:

• Analyzing ceramides and fatty acids

• Detection of pesticides or insecticides in food and water

• Analyzing the dye composition of fibers in forensics

• Assaying the radiochemical purity of radiopharmaceuticals

• Identification of medicinal plants and their constituents

Thank You