Question

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

These stains will readily give up a hydroxide ion or accept a hydrogen ion, which leaves the stain positively charged. Since the surface of most bacterial cells and cytoplasm is negatively charged, these positively charged stains adhere readily to the cell surface. After staining, bacterial cell morphology (shape and arrangements) can be appreciated.

Naphthol Blue Black is Bio reagent.for electrophoresis.Naphthol Blue Black has been used for the staining of western blot membrane for detection of all protein that are transferred to the membrane.^[1Sructure : Molecular formula : C22H14N6Na2O9S2;. Molecular Weight: 616.487 g/mol

In this experiment, lipids is analyze from the egg yolk. The yolk makes up about 33% of the liquid weight of the egg; it contains approximately 60 calories, three times the caloric content of the egg white. Egg yolk contains trioleine, cholesterol palmitate and phosphatidylethonolamine, The most polar of the three elements is the second element. It hardly moves up the TLC because of the presence of several polar groups; phosphate groups, amine groups and several oxygens , and cholesterol palmitate being the most non polar. In this practical 'thin layer chromatography' is used. TLC is a form of adsorption chromatography where the substances we wish to separate are absorbed onto yhe 'thin layer' and allow a solvent to pass through the thin layer matrix. In this practical, TLC is used to separate lipids present in egg yolk. Following separation of the lipids two lipid visualisation techniques are used - one which detects lipid in general and one which detects sterols. In order to identify the different lipids, known standards are required. Using the pattern of spots on the TLC plate an attempt was made to deduce the composition lipids in egg yolk. Lipids are separated on the basis of hydrophobicity. Eggs contain cholesterol and phospholipids. The least hydrophobic molecule moved to the top of the TLC plate. The most hydrophobic molecule, Cholesterol palmitate, did not move up the solvent front. Cholesterol palmitate is not a polar molecule. Hence, cholesterol palmitate did not dissolve in the solvent front, and so could not be moved up the TLC plate. Egg yolk is 23% palmitic acid, the egg yolk did not move up the solvent front due to the percentage of non-polar cholesterol palmitate it contains. Linolic acid is another molecule present in egg yolk. Egg yolk is 16% linoleic acid. Linoleic acid is a carboxylic acid and so it is a

1. polar molecule, hence linoleic acid was carried along the solvent front on the TLC plate. Egg yolk displays hydrophobicity as it's non-polar components are dominant over the polar parts. This is why egg yolk was not carried up the solvent front. Lipid Molecular Structure EXPERIMENTAL A. Materials Needed TLC Solvent Mixtures: Petroleumether:methanol:NH4OH Petroleumether:methanol:water (65:25:4) (65:25:4)
2. 3. Ninhydrin Spray Iodine Crystals B. PROCEDURE Equilibrate the TLC solvent mixtures in two separate beakers: (a.) 65:25:4 (v/v/v) petroleum ether:methanol:water (b.) 65:25:4 (v/v/v/) petroleum:ether:methanol:NH4OH. Place two clean TLC plates on the hot plate with silica side up for approximately 3minutes to reactivate silica.Remove TLC plates from heat. Spot the extract (from the extractin of total lipids from chicken egg yolk) at least 1cm from the edge of the TLC plates.Develop the plate in the first solvent mixture 65:25:4 (v/v/v) petroleum ether:methanol:water.Remove the TLC plates when the solvent front is almost ¼ inch from the top and transfer them to the second solvent mixture 65:25:4 (v/v/v) petroleum ether:methanol:NH4OH. Put I2 crystals in a separate beaker and saturate the container with I2 vapor for 5 minutes. Transfer the TLC plates in the beaker with iodine until spots appear. Place the plates on a hot plate to remove excess I2. Completely spray the TLC plates with ninhydrin. Place them on the hot plate for 1minute. A blue-violet colorization indicates the presence of a-amino acids in the ninhydrin test.Completely spray the plates with phosphorus. Place them on the hot plate for 30 seconds. Phosphorus containing compounds will appear as blue spots on white background. Additional heating will char unsaturated compounds.