THIN LAYER CHROMATOGRAPHY ANALYSIS PROCEDURE
Thin Layer Chromatography
Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle.
They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates.
Thin layer chromatography is done exactly as it says - using a thin, uniform layer of silica gel or alumina coated onto a piece of glass, metal or rigid plastic. The silica gel (or the alumina) is the stationary phase. The stationary phase for thin layer chromatography also often contains a substance which fluoresces in UV light - . The mobile phase is a suitable liquid solvent or mixture of solvents.
Producing the chromatogram
A pencil line is drawn near the bottom of the plate and a small drop of the extracted oleoresin is placed on it. Any labeling on the plate to show the original position of the drop must also be in pencil. If any of this was done in ink, dyes from the ink would also move as the chromatogram developed.
When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a covered beaker. A mixture of cyclohexane , chloroform and acetic acid in the ratio of 6:3:1 is used as a solvent here. It is important that the solvent level is below the line with the spot on it. The reason for covering the beaker is to make sure that the atmosphere in the beaker is saturated with solvent vapour. To help this, the beaker is often lined with some filter paper soaked in solvent. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the plate.
As the solvent slowly travels up the plate, the different components of the chilli oleoresin travel at different rates and the mixture is separated into different coloured spots. The solvent is allowed to rise until it almost reaches the top of the plate. That will give the maximum separation of the chilli components for this particular combination of solvent and stationary phase.
Position of the spot on a thin layer plate.
TLC plate showing distances traveled by the spot and the solvent after solvent front nearly reached the top of the adsorbent.