Question

Explain thin layer chromatography and its utility in determining the components of a mixture. Be sure to briefly explain how TLC works as well as what and how the visualization techniques work.

Answer 1

Thin layer chromagraphy is a chromatographic technique used to seperate and identify the compounds in a mixture. This used the different binding affinities of the compounds in the mixture to the stationary phase and the mobile phase.

In TLC the stationary phase is coated on glass plates, metal( aluminium) plates or plastic films as a thin layer. The most commonly used substances for stationary phase are silica gel, alumina and cellulose powder.The adherence to the plate is ensured by adding a binding agent like calcium sulphate into the stationary phase. If the visualization is done under uv a fluorescent compound is also incorparate into the stationary phase. The prepared TLC plates are also known as chromaplates.when handling these chromaplates care should be taken not to touch the face of the plate to prevent damaging the stationary phase and also to prevent contaminations. They should always be handled using the edges.

The sample is spotted about 1cm above the edge of the plate. This 1cm margin should always be drawn with a pencil. Sample is spotted using a small capillary. After the spot is properly dried the plate is placed in a closed jar containing the solvent or the mobile phase. Before placing the TLC plate the jar should be allowed to saturate with the mobile phase used. The stationary phase and the mobile phase are chosen based on the compounds in the sample. When the solvent reached about 1cm below the upper edge the plate should be taken out from the jar and the solvent front should be marked with a pencil.

The mobile phase rise along the TLC plate due to the capillary action. The compunds that are soluble in the mobile phase will rise along with the mobile phase and the compounds that are bound strongly to the stationary phase will not rise with the mobile phase. For example if the compound is polar and stationary phase is also polar, and a non polar mobile phase is used, that compound will not move with the mobile phase as it is strongly bound to the stationary phase. Because of this differential distribution, the compounds in the mixture are seperated. If the compounds in the mixture are known sample can be run along with standard samples of the known compunds and this can be used in identification.

The identification and the analysis of the seperated compounds is done by using the retention factor(R_f).

R_f= Distance traveled by the compund/ Distance traveled by the solvent front

If the compound in the sample is same as the standard both will have the same R_f.

There are various visualizaton methods used in TLC.

• using plates incorparated with fluorescent compounds can be visualized under uv of wavelenght 254nm.
• exposing the plate to iodine or bromine vapour.compounds with unsaturations can be seen using this as unsaturated compounds form complexes with iodine and bromine.
• spraying pH indicators. Compounds with acidic or basic properties can be identified using this.
• spraying general locating agents
• spraying specific locating agents. e.g:-Dragendorff spray for alkaloids.

The spots visualized using these methods have to be marked with a pencil and the R_f value is calculated for analysis.