By Sebastian on Wednesday, March 31, 2004 - 11:55 pm:
I am developing a method for the cleanup of serum in order to extract very lipophilic compounds (chlorinated pesticides, PCBs, plastizicers).
The SPE-extract will be further cleaned up on a HPLC system.
My question is: When i have conditioned the SPE- cartridge (Oasis HLB) with methanol and water, and have applied the serum, there is a washing step before the elution of the desired compounds.
The washing is done with water with 5% methanol (some texts also uses 5% acetone). Methanol is suggested by Waters, the manufacturer of the cartridges.
- Why is methanol added to the water?
- Would it be a problem to add acetone instead?
- How does the addition of a modifier to the water affect the recovery of apolar compounds?
- Would it make a big difference if i changed the composition to 10 or 15%?
- Is it necesary to add a modifier at all? and if so why?
Thank you for any replies!
/Sebastian
By Uwe Neue on Thursday, April 1, 2004 - 03:37 pm:
The first washing step in the standard Oasis HLB serum extraction protocol is designed to remove salt, sugars, proteins and other very polar interferences. You may get away without it, but why bother?
The addition of a small fraction of organic solvent to the water will not affect the recovery of the very lipophilic compounds. As a matter of fact, you might get away with an even stronger wash solvent.
The reason for the wash is to clean up your sample. A hydrophobic sorbent does not only retain your analytes, but all kinds of other small molecules in your plasma. A wash with a solvent containing more organic will improve the sample clean-up. You just do not want to make the wash strong enough such that some of your analytes are eluting.
Finally: With very lipophilic analytes, you may consider a stronger eluting solvent than the standard methanol. Acetone is a better choice (more hydrophobic), but if you still do not get 100% recovery, I would think about a still stronger eluent.