I have a series of N-acetylated peptides in the form of Ac-AnK-OH, where n = 2-6. They are of decent purity ~80% but still have some residual TFA and smaller contaminants that make it difficult for mass spectrometry. I was considering using prep-HPLC to purify these peptides but am wondering if it will have any absorbance at all.
Id like to hear any experiences, techniques, tips&tricks, etc...If it doesn't, are there any other methods used to purify these types of peptides that I can be referred to?
Thanks!
I believe I read somewhere (I've been reading about capillary electrophoresis) that the peptide bond absorbs ~210nm though almost everything does. Otherwise you'd have to label it.
In addition to the good ideas above, one could obtain a universal detector:
MS, corona discharge, or evaporative light scattering.
Refractive index is old technology: one cannot run gradient elution with RI.
Hi Patrick, I do agree with the others. Wavelength at 210 to 220 nm should work with peptides if you will use high enough concentrations. The quality of your clean up by RP-HPLC will depend on column material and the interaction of the specific peptide (polarity).
HPLC with UV and MS always uses 214 nm to detect peptides (absorbance of the peptide bond), 280 nm is standard used for proteins.
I use 214 nm for many years. However: solvents (acetonitril, water, TFA) must be very pure!
How about just tracking the conductivity? Your salts and acid will have much larger conductivity then the fraction of the protein (but, I do work with larger proteins). I usually see a dip in conductivity when the protein is coming out and a sharp spike when the salts are coming out. My AKTA FPLC comes standard with a pH/conductivity probe. Might be worth trying if your system has the same.
Agree with previous responses. Fix the wavelenght of your UV detector at 215nm, characteristic of peptide Bond absorbance. The absorbance will increase following the lenght of your peptides and their concentrations.
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