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

A. Sheppard mbasd at
Mon Jan 16 04:37:57 EST 1995

Mr. P.A. Sansom (psansom at wrote:
: 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 
: synthetic, but sequence analysis and mass analysis show it to be pure, ie 
: there is only one main species present. Also, I've had three separate 
: preparations made at two different sites, and the effect is seen in all of 
: them!

: Trypsin treatment cleaves AIH from the C-terminal resulting in a single peak
: corresponding to DHLSDNYTLDHDR and a small double peak corresponding to 
: AIH indicating that the variation lies in this AIH region.

Possibly a racemisation during the synthesis of the tripeptide? Histidine
is quite prone to racemisation, and having it at the C-terminus can cause
particular problems: the racemisation would probably occur right at the first
step when the amino acid was attached to the resin.

Solution: get a better prep. HPLC column or get your peptide suppliers to
analyse the peptide resin at the tripeptide-resin and histidine resin stage.
If this reveals the histidine has racemised, a different resin or even a
fragment coupling approach may be required.

Good luck


Dr. Andrew Sheppard
Project Leader, Drug Design and Discovery
Ferring Research Institute             Tel: 011 44 1703 760855
Southampton University Research Centre Fax: 011 44 1703 766253
Chilworth, SOUTHAMPTON, SO1 7NP, U.K.  e-mail:mbasd at

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