Chromatography Forum

I don't know if this is a relavant post to this board, but I'm looking for some information on SPE preparation for HPLC analysis.

I'm trying to separate peptide from acetate and TFA. I thought the peptide would be the difficult issue, but I can now retain and wash the peptide with close to 100% recovery. The difficulty is with the acetate and TFA. I had thought they would come off either during loading or the first wash, but I seem to get acetate/TFA in the eluates of all steps.

So, now I want to focus on the acetate/TFA and then work what I know about the peptide into the method afterwards. Any ideas?

Ultrafiltratiion, or SEC could be a simple alternative (if your peptide is big enough).

OK. Please describe your method in more detail. You should be able to do what you want to do without too much trouble.

What SPE are you using, how do you load the sample, what do you do to wash off the acids, how do you get the peptide back off?

I wouldn't use ultrafiltration as you might have some significant losses. SPE should work especially as you already see good recoveries for your peptides. Extensive wash followed by elution with water-methanol or water-acetonitrile and subsequent evaporation should do the job...

There are centrifuge ultrafilters specially made for peptides and proteins which do not show any more loss than a SPE catridge for most samples. Even sticky proteins can be tought to behave with the proper medium, usually much easier to find than correct chromatographic conditions.