ColumnPurificationofDemethylatedSphingomyelin

Packing column:

1) To 20 g of 100-200 mesh Bio-Sil A silica gel add 80 mls of chloroform.

2) Place a small portion of glass wool at the base of the column.

3) Pour gel solution into column and use a stir bar to press out any bubbles in the glass wool.

4) Slowly drain the chloroform while stirring to prevent bubbles from being present in the packed gel.

5) Add 20 mls more chloroform to recover the remaining portion of silica gel and also pour into column.

6) Leave a small portion of chloroform solution above the level of packing.

Column run:

1) Load sample dissolved in chloroform just above the packing.

2) Allow sample to permeate the gel and start eluting, collecting fractions.

3) Elute with:

a) 100 mls chloroform

b) 200 mls methanol-chloroform (1:20)

c) 200 mls methanol-chloroform (1:9)

d) 200 mls methanol-chloroform (1:5)

e) 200 mls methanol-chloroform (1:4)

f) 800 mls methanol-chloroform (1:3)

g) 200 mls methanol-chloroform (1:3)

4) Dry down all fractions on the rotovapor using house vacuum and resuspend in a few mls