SPE-silica-columns

Solid phase extraction (SPE) of complex lipid extract on silica column

1. Dissolve lipid extract in 100 % chloroform

2. Use small silica columns (e.g. Strata SI-1 Silica (55 µm, 70 Ǻ)), 500 mg or 100 mg silica, or self-made Kieselgel 60
    column in a Pasteur pipette with glass wool or in a glass column with frit.

3. Equilibrate silica column by washing with 3 mL or 15 mL of 100 % chloroform for 100 mg or 500 mg columns,
    respectively.

4. Apply lipid sample in chloroform, collect flow through.
    Elute with 3 mL or 15 mL of 100 % chloroform for 100 mg or 500 mg columns, respectively. If you have a green extract
    (leaf), the eluate should turn from green to colourless before you start with step 5. Combine all chloroform phases in
    one tube. This fraction contains the neutral lipids: triacylglycerol, diacylglycerol, free fatty acids, free sterols, sterol esters,
    fatty acid phytyl esters and free phytol. Neutral lipids can be further fractionated using SPE as described here (SPE of
    neutral lipids).

5. Elute with 3 mL or 15 mL of acetone/isopropanol (1:1, v/v) for 100 mg or 500 mg columns, respectively.
    This fraction contains the “glycolipids” such as plant galactolipids (MGDG, DGDG) and sulfolipid (SQGD),
     hexosylceramides (GlcCer, GalCer), free sphingobases/long chain bases, ceramides, sterol glucosides and acylated
     sterol glucosides.
 
6. Elute with 3 mL or 15 mL of 100 % methanol for 100 mg or 500 mg columns, respectively.
    This fraction contains phospholipids including PS, PI, PG, PE, PC, some lyso-phospholipids and sphingomyelin from
    animals.

7. Elute with 3 mL or 15 mL of methanol/water (1:1, v/v) for 100 mg or 500 mg columns, respectively.
    This fraction contains lyso-phospholipids (e.g. lyso-PC).

Solid phase extraction (SPE) of neutral lipids on silica column
Modified from cyberlipid.org

1. Dissolve neutral lipids in 1 mL of 100 % n-hexane.

2. Use small silica columns (e.g. Strata SI-1 Silica (55 µm, 70 ?)), 500 mg or 100 mg silica, or self-made Kieselgel 60
    column in a Pasteur pipette with glass wool or in a glass column with frit.

3. Equilibrate silica column by washing with 3 mL or 15 mL of 100 % n-hexane for 100 mg or 500 mg columns, respectively.

4. Apply neutral lipids in n-hexane, collect flow through.
    Elute with 3 mL or 15 mL of 100 % n-hexane for 100 mg or 500 mg columns, respectively.
    This fraction contains hydrocarbons and squalene.

5. Elute with 3 mL or 15 mL of hexane/diethylether (99:1, v/v) for 100 mg or 500 mg columns, respectively.
    This fraction contains the fatty acid phytyl esters, sterol esters, waxes and fatty methyl esters.

6. Elute with 3 mL or 15 mL of hexane/diethylether (95:5, v/v) for 100 mg or 500 mg columns, respectively.
    This fraction contains triacylglycerol and tocopherol.

7. Elute with 3 mL or 15 mL of methanol/water (1:1, v/v) for 100 mg or 500 mg columns, respectively.
    This fraction contains lyso-phospholipids (e.g. lyso-PC).