Dear all
I am working right now with Supel™-Select SPE HLB cartridges to separate hydrophylic from hydrophobic peptides from a mixture. And it seems not to be working properly.
Experiment Details
The initial sample is 0.1 g diluted in 1 ml
The protocol followed is the one recommended for the cartridges: 1. 100% methanol, 2. water, 3. sample, 4. 10% methanol, 5. 50% methanol to elute the second fraction
The process is speed up by a vacuum pump
If you are working with the same or similar cartridges. Do you think we shouldn't use the vacuum because we are eluting everything fast? Do you think we are doing something else wrong?
I would really appreciate all the comments
Thanks a lot
The SPE protocol you described seems generic and should work. The question is how fast you were eluting the compound(s) from the SPE cartridge. We found that 1 drop per second speed is a good starting point. Do not apply too much vacuum or the SPE process will fail. Depending on your compound(s) you might have to increase the % organic during the elution steps or even change to a stronger solvent. Sometimes the addition of a small amount of acid/base might also help.
Besides flow rate think of polarity. Maybe 10% Metanol is too much to wash.
Try washes with increasing percentage of organic solvent on identical replicate samples. You should start with water alone as the wash solution.
Since HLB has to retain ionezed analytes more strongly than silica-based-reversed-phase sorbents, think of a pH solution when washing.
In the three previous comment should be the key for a successful fractionation. Flow should not be too fast and 10% MeOH is probably too much organic for a whash solvent. As an example you can see http://www.covance.com/content/dam/covance/assetLibrary/posters/liaaps12spdf.pdf
Thank you all for your contributions...I'll repeat it again and let's see what happens...Thanks a lot
Dear Aurea,
Thank you very much. We did achieve the separation of the peptides at the end by acidifying the peptides with 0.1% TFA and without pump in pre-packed Oasis HLB tubes.
We could actually see that the tubes were cleaned after the process, unlike the previous standard protocols used.
Thank you very much for your comment
Regards
Maro
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