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Solid phase extraction (SPE)

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Please make sure that your samples are free of detergents, PEG, plasticizers etc. We recommend that you extract your samples at your institution and ship them to us at ambient temperature.

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

  1. Dissolve lipid extract in 100 % chloroform. Note: Depending on the supplier, chloroform can be contaminated with triacylglycerols, we therefore strongly recommend to discuss the use of chloroform with us previous to the sample preparation, if you want to analyze triacylglycerols.

  2. Use silica columns (Macherey and Nagel CHROMABOND SiOH (45 µm)), 6 ml/500 mg or 1 ml/100 mg). Note: silica columns from other suppliers can be contaminated with triacylglycerols, diacylglycerols and free fatty acids, we therefore strongly recommend the Macherey and Nagel columns for these lipid classes.

  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 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.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 silica columns (Macherey and Nagel CHROMABOND SiOH (45 µm)), 6 ml/500 mg or 1 ml/100 mg). Note: silica columns from other suppliers can be contaminated with triacylglycerols, diacylglycerols and free fatty acids, we therefore strongly recommend the Macherey and Nagel columns for these lipid classes.

  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).