Techniques in Biotechnology Chapter 2: Thin Layer Chromatography (TLC)

Principle of TLC

• The principle of TLC is based on the difference in the partitioning behavior of the components of a mixture between the stationary phase and the mobile phase.

Separation and Visualization

• A less polar compound moves faster and farther up the TLC plate, while a more polar compound moves slower and stays closer to the starting point.
• Individual separated compounds are visualized in TLC by using a chemical reagent or by exposing the plate to iodine vapor or ultraviolet light.

Stationary Phase

• The solid phase used in TLC is usually a thin layer of silica gel or alumina on a glass plate or plastic sheet.
• The stationary phase is responsible for separating the components of a mixture based on their polarity.

Rƒ (Retardation Factor) Value

• The Rƒ value is calculated by dividing the distance traveled by the compound by the distance traveled by the solvent front.

Main Purpose of TLC

• The main purpose of using TLC is to identify, separate, and quantify the components of a mixture.

Role of Stationary Phase

• The role of the stationary phase is to separate the components of a mixture based on their polarity, allowing them to be visualized and characterized.

Stationary Phase Materials

• Silica gel, alumina, and cellulose can be used as the stationary phase in TLC.

Mobile Phase

• The mobile phase in TLC is a solvent that moves up the plate, carrying the components of the mixture with it.

Applications of TLC

• TLC can be used to determine the purity of a substance, identify the components of a mixture, and monitor the progress of a reaction.