1) Prepare a C-18 reverse phase prep-sep cartridge by washing with: a) 10 ml of methanol b) 20 ml of chloroform-methanol (2:1) c) 10 ml of methanol d) 10 ml of methanol-1.6 M sodium acetate (1:1) 2) Apply sample in 2 ml of methanol-1.6 M sodium acetate (1:1). 3) Collect wash through and apply twice more to the cartridge. 4) Wash cartridge with 40 ml of water. ---> This actually washes away the salt from the glycosphingolipids. 5) Elute acid glycosphingolipids with: a) 2 ml of methanol b) 10 ml of chloroform-methanol (1:1) 6) Pool the two eluates and dry lipids down. 7) Resuspend in a minimal volume of chloroform-methanol (1:1) and spot on TLC. 8) Develop TLC in chloroform-methanol-0.22% calcium chloride (60:35:8).
