- Egg yolk is collected and mixed well, to this 150ml of cold acetone and allowed to stand for 15 mins.
- 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.
- The extract is evaporated to dryness the residue is taken up in a small volume of petroleum ether (25-30 ml)
- The phospholipids are re-precipitated with the addition of cold acetone (200ml)
- The precipitate is collected and dried.
- The TLC chamber is filled with solvent chloroform – methanol – acetic acid - water [25:15:4:2] and is kept tightly closed.
- Dried precipitate is taken in an eppendroff tube and to this petroleum ether is added, Mix well and let it stand
- Phospholipid gets dissolved in ether and clear liquid phase is attained.
- Transfer this liquid phase to a fresh eppendroff tube.
- Using a capillary tube, spot samples on the TLC plate.
- When the sample gets dried transfer the plate into TLC chamber for 15 mins (till the solvent moves 3/4th of the plate)
- The plates are then taken out and placed in a chamber which is saturated with iodine vapors for about 2 mins.
The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.