Contributor: Suprya JayadevDate: November 10, 1993
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).
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