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

Peter J. Neame pneame at
Mon Jan 23 19:30:52 EST 1995

Malcolm Moos Jr. (moos at wrote:
: mbasd at (A. Sheppard) wrote:
: >
: > Mr. P.A. Sansom (psansom at wrote:
: > : Single peptide is 2 peaks on RP-HPLC. Why?
: > 
: > :Also try running your separation at elevated temperature (>50°) to eliminate
: the possibility of conformational isomers. I routinely dissolve peptides
: in guanidine before HPLC analysis to help with this problem and make
: sure the sample is completely dissolved.

Dissolving them in guanidine doesn't do much to some ultra-stable 
structures. Also, I believe that secondary structure can be reasonably 
happy at pH 2 (the pH of a typical TFA-based HPLC solvent). However, this 
peptide which is the N-terminal of a cartilage protein, doesn't have too 
much structural stuff. It just sort of hangs out in the breeze. So I'd 
say this is definitely racemization. It's nailed down to three amino acids!

Peter Neame
University of S. Florida.
Biochem + Mol. Biol.

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