Single peaptide is 2 peaks on RP-HPLC. Why?

John E. Fox altabios at bham.ac.uk
Tue Jan 17 09:23:00 EST 1995


In article <3f9bof$i21 at mserv1.dl.ac.uk>, psansom at hgmp.mrc.ac.uk (Mr. P.A. Sansom) says:
>
>Single peptide is 2 peaks on RP-HPLC. Why?
>
>Background
>I'm using C18 reverse phase HPLC (TFA and acetonitrile gradients) to 
>separate fragments of a digested peptide. The problem is that the starting 
>peptide (16 amino acids, sequence NH2-DHLSDNYTLDHDRAIH-COO) detected at 
>206nm runs as two equal height overlapping peaks, not separable. It is 
>


The problem may be due to racemisation of the His during the synthesis.
C-terminal His is notorious for doing this. This would explain the correct
mass spec. Specify that the synthesis should be done with FMOC His trityl resin.

If you can, use C-term amide or extend/reduce the pepide by one amino acid,
or better still get it made by Alta Bioscience.



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