Thin Layer Chromatography - The Assay of Sulfamethazine in Pork Carcasses 3
Five hundred gram of finely ground palm kernel (appropriately sampled) were slurried with 750 ml of water in a blender at high speed for 3 min. A 100 g portion was then extracted by blending with 240 ml of acetone and filtered through a Whatman No. 1 filter. 5 ml of the crude extract was mixed with 5 ml of methanol, 1 ml of 20 %w/v lead acetate solution, 1 g of Celite and 63 ml of water. The lead salt was used to precipitate colloidal contaminants from the sample. The mixture was then passed through a solvated PH bonded phase cartridge at about 10 ml/min. (PH cartridge 608303 was solvated by treating with 10 ml of methanol followed by 10 ml of water). The cartridge was then washed with 10 ml of water and the aflatoxins eluted from the cartridge with 3 ml of chloroform.
The extract was dried by passing it through a tube packed with anhydrous sodium sulfate and the dry extract collected in an 8 ml vial. The solid aflatoxins were obtained by evaporation at 45˚C in a stream of nitrogen. The HPTLC plates (aluminum backed, 20 x 20 cm, Merk 5547) were immersed in methanol for 1 hr to remove any adsorbed material, dried for 5 min at 100˚C and stored in a dessicator. The sample extract was dissolved in 300 ml of a benzene:acetonitrile mixture (98:2). 5 ml aliquots, occasionally interspersed with standards, were applied to the plate in the absence of light with an auto sampler. Sample spots were 5 mm apart along the top edge of the plate and one 10 x 20 cm plate could accommodate 30 sample spots and three standards along the 20 cm edge. Edge effects were eliminated by removing a strip of silica gel, 3 mm wide, along the plate edge parallel to the direction of development.