Fractionation of Phospholipid by Thin Layer Chromatography (TLC)

Phospholipids are group of lipid molecules containing phosphorus. The bases contain are choline, ethanolamine, inositol and serine phospholipids are frequently found in association with proteins and constitute the essential part of all cell membranes. The phospholipids are amphoteric nature because of the hydrophilic and hydrophobic part and which is of great importance.

Solvent used for Fractionation: chloroform – methanol – acetic acid - water [25:15:4:2]

Other Requirement : Ice cold acetone, Choloroform - Methanol mixture

Procedure: Isolation of phospholipid from egg yolk.

1. Egg yolk is collected and mixed well, to this 150ml of cold acetone and allowed to stand for 15 mins.
2. The precipitate is collected by centrifugation. Then the precipitate is extracted with 500ml of chloroform – methanol mixture (2:1) for 3-4 hours at room temperature.
3. The extract is evaporated to dryness the residue is taken up in a small volume of petroleum ether (25-30 ml)
4. The phospholipids are re-precipitated  with the addition of cold acetone (200ml)
5. The precipitate is collected and dried.

Thin Layer chromatography for Phospholipids

1. The TLC chamber is filled with solvent chloroform – methanol – acetic acid - water [25:15:4:2] and is kept tightly closed.
2. Dried precipitate is taken in an eppendroff tube and to this petroleum ether is added, Mix well and let it stand
3. Phospholipid gets dissolved in ether and clear liquid phase is attained.
4. Transfer this liquid phase to a fresh eppendroff tube.
5. Using a capillary tube, spot samples on the TLC plate.
6. When the sample gets dried transfer the plate into TLC chamber for 15 mins (till the solvent moves 3/4^th of the plate)
7. The plates are then taken out and placed in a chamber which is saturated with iodine vapors for about 2 mins.

 Retention Factor (R_f)
The retention factor, or R_f, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.

_{Rf =} Distance Travelled by compound / Distance Travelled by the solvent

The Rf Values can be calculated from the above equation.