Thin Layer Chromatography - Determination of the Platelet-Activating Factor and other Phopholipids in Human Tears 2

 

Tear samples were obtained by introducing 40 ml of physiological saline solution into the inferior conjunctival sac. After three gentle eye blinks, a 2 cm diameter circle of filter paper was placed near the inner canthus for tear collection. The filter paper was then soaked in 500 ml of chloroform-methanol-water mixture (6:69:50 v/v). The liquid was then passed through a diatomite column to remove proteins, sugar and polar inorganic substances and the lipids were then eluted with 6 ml of a chloroform-methanol mixture (95:5). The eluate was evaporated to dryness under vacuum and the residue dissolved in 50 ml of chloroform, 20 ml samples of the solution were used to spot the TLC plate.

 

A high performance silica gel plate was employed (HP-K, Whatman) which was soaked overnight in a methanol water mixture (1:1 v/v) and then dried for 1 hr at 120˚ prior to use. The plate was developed in two dimensions; first with a hexane-diethyl ether mixture (4:1 v/v) for the removal of lipids other than phospholipids; second the plate was then developed at right angles with a chloroform-methanol-water mixture (65:35:7 v/v) to separate the phospholipids. After drying, the plate was sprayed with phospholipase C (PL-C from Bacillus cereus) and then alkaline phosphatase (Al-P from human placenta) to hydrolyze the phospholipids.