Once you remove the organic phase from your sample, do you aliquot a defined amount from all your samples and then dry it down and reconstitute or do you dry you sample in its entirety and the reconstitute?
I understand if you do the former, you have less lipid recovery, however,you will still be able to compare your biological and technical replicates, correct. Meaning that your data will still be valid even though the total mass of the lipids will be less across all your samples
In this case, it is a modified
B&D but my question could be applied to either method.
The same here. We dry the B&D organic phase completely using nitrogen gas. Then reconstitute in a specific volume of chloroform:methanol (2:1). We have never seen any sample loss because of this.
However for total lipid mass, we take two small aliquot of this reconstitute and we do duplicates measurements in little aluminum weighting dishes. We discard these aliquots so we do loose some sample.
An alternative, if you have enough lipids in your extraction, you can weight a a glass tube transfer your extraction in it, dry it completely and then weigh it again to calculate your lipid mass. Then re-suspend your lipid in your favorite solvent with out loosing any sample.
Hope this helps!
Yes, you will have less lipid recovery if you take an aliquot rather than use the entire sample. This is trivial in making the decision though, and you should be accounting for the fraction of lipids that you remove in an aliquot with an internal standard or simply algebraically. What you need to ask yourself is, what is the preferred concentration of lipids in the GC vial prior to analysis, and what fraction of your total lipid extract do you need to achieve this? Because lipids require derivatization (at least the Blight and Dyer method does with BF3), I find it is always best to only use an aliquot if possible. Because this final phase is destructive to your sample, if there is an error with the GC/MS you can always get another aliquot and re-do. If there are final concentration concerns you can always use a GC vial insert and re-dilute up to a small volume like 100ul after drying down
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