Desalting Acidic Glycosphingolipids

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).