We work with lipids in milk and usually I prefer using SPE. Can you recommend other methods to isolate (preparative) monoglycreides from diglycerides and from triglycerides?
When I was a postgraduate student I used to fractionate neutral acylglycerols such as TAGs, sn-1,2-DAGs and 2-MAGs (obtained both from total lipid extract and as a result of lipase hydrolysis of TAGs) using the preparative thin-layer chromatography on Merk's silica gel plates preimpregnated with boric acid (to avoid acyl migration) and 2'7'-dichlorofluoresceine to visualise zones with separate NAGs classes under UV light. It is really convenient and reliable method.
Prior lipid extraction (Folch or Bligh Dyer) and SPE on a normal phase (silica) column gives excellent separation of TAG/DAG and MAG. Preparative TLC will also work but the loss of critical MAG species like 2-AG is higher (at least in our experiments)!
The extraction step is no problem, but i want spe an we have used aninopropyl, idol and silica column and we still have crossover or cryss contaminants
When you extract the milk according to Folch and use the organic phase for SPE I would suggest a selfmade silica SPE column (can be used as gravity flow column w/o vacuum). However you can also used a prepacked cartridge.
The elution of gross TAGs and cholesteryl esters should be done with pure CHCl3 followed by a CHCl3/MeOH (99:1, v/v) step to elute residual TAG/DAG. The next step using CHCl3/MeOH (9:1, v/v) yields the MAG fraction. We always analyze the lipids with LC/MS and this fraction is TAG/DAG free.
Ok,
We have been working With standards of mag, dag and tag. And we use a spe robot and prepacked cartridge
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