Question

During lipid metabolism experiment, several reagents were used, what   is their role of the TLC developing reagent (petrolum ether:ether:glacial acetic acid)?

Answer 1

Thin layer chromatography is a separation technique which is based upon partitioning of the components of the mixture between the stationary and mobile phase based upon their relative polarities. Its performed on a sheet of aluminium, plastic or glass.

After the sample has been applied on the plate, a solvent mixture I.e., mobile phase, migrates up the plate by capillary action. Because different analyte ascend the TLC plate at different rates, separation is achieved.

Stationary phase in a standard TLC experiment is the silica.

Mobile phase may vary, and as in the question its a mixture of Petroleum ether: ether: glacial acetic acid.

The polarity index of the solvent components are -

• Petroleum ether = 0.1 (most non polar)
• Ether = 2.6 (slightly non polar)
• Glacial acetic acid = 6.2 (most polar)

Since, the aim was to separate a mixture of polar and non polar components of the lipid mixture obtained during the lipid metabolism experiment. Thus, those components which are non polar would dissolve in petroleum eteher and migrated sp the tLC plate while those which are less non polar or polar would dissolve in ether and glacial acetic acid respectively which would migrate upwards.

After the separation is complete, the migration of lipids on the plate can be detected using iodine.which reacts with the double bonds in fatty acids, giving the lipid content spots.