Question

For TLC, How does the staining procedure work? What is Napthol Blue-Black and how does it interact with lipids? TLC analysis of egg lipids

Answer 1

At times, once the TLC has been developed, the observation/location of the separated components become unclear/invisible due to the fact that most of the organic compounds are colourless. Hence, they require visualisation. This is acquired using visualizing agents, which for lipids happen to be Naphthol Blue-Black.

In this case, Naphthol Blue-Black is used for staining process which is an amino acid staining dye, i.e. both the polar as well as non-polar regions of Napthol Blue-Black. These regions interact with the compound thereby forming dipole and hydrogen-bonding interactions with the lipid. Specifically, the polar interactions occur between polar region of the dye and active hydrogens present in the amino acids. These result into the chromophores absorbing light such that a visible colour is emited, thereby making the compound visible.

In case of lipids, the separation occurs on the basis on one's hydrophobicity, i.e. relative absorption of the hydroxyl groups attached to the silica atoms. The solubility of the component of the lipid with either stationary phase or mobile phase, or. their interaction with the respective phase determines to what extent they travel in it and hence their spearation. The most hydrobphobic one travels the least whereas the least hydrophobic molecule travels the most as it has highest affinity for mobile phase.

Eggs contain cholesterol and phospholipids. Cholesterol being non-polar is the most hydrophobic component. Doesnt dissolve in the solvent front, and so has more affinity for stationary phase. Egg yolk also contains linoleic acid (polar molecule), hence is carried along the solvent front. In general, the non-polar components in egg yolk dominates over the polar componets and therefore egg yolk doesnt travel along with the solvent front.