Peptide HPLC Sample Prep - (Apr/22/2013 )
I am trying to purify a bacterially expressed peptide using RP-HPLC. In order to use HPLC, I dialyzed the peptide against pure water and lyophilized it. Because it's just pure water with no salt, the peptide precipitates. When I try to solubilize it in water to load on the HPLC, the peptide is still precipitated. I was able to use 50% MeOH to solubilize it for MS. But I fear that MeOH would prevent my peptide from interacting with the RP column. I suppose this happens quite a bit, though I am not sure what people use to overcome the solubility issue? I've read about adding TFA, but only to a 0.07%? I am currently re-expressing the peptide. Would small amount of TFA solubilize the peptide? How much TFA can I add?
Thanks a lot for your time
from the handbook on hic and rp chromatography from ge healthcare:
It is important to maintain sample solubility throughout the loading process and during separation in
order to avoid precipitation on the column.
If back pressure increases significantly this may be a sign that sample is precipitating on the
column. Recheck sample solubility in eluent A. A low percentage of organic modifier in eluent A,
for example, 5% acetonitrile, helps to overcome solubility problems without disrupting the
If there are problems with solubility when the sample is dissolved directly in eluent A, add formic
acid or acetic acid (0.1%) to increase solubility. Keep the sample volume small compared to the
column volume to avoid any interference by these additives. If large sample volumes are applied
these additives will be seen as additional peaks eluting in the void volume after injection.
Ensure that sample is at the same temperature as solutions, columns and chromatographic
Do not overload the column as this can also cause precipitation
you can download this handbook from this webpage.
We always resuspend our lyophilized peptides in 0,1% TFA in water before nano-HPLC and it works quite well.
For hydrophobic peptides 5% acetonitrile can help but is generally not necessary.