Chromatography Forum

I have a peptide with a cis/trans isomerizing bond between a tryptophan and proline. I would like to separate the cis/trans isomers on a standard HPLC column (i.e. C18)- any suggestions for an ACN gradient, stationary phase, etc?

This separation isn't limited to reversed-phase chromatography. Proline can interconvert between two conformations. Normally this interconversion is fast relative to the time scale of chromatography, and one gets a single peak that's the time-weighted average. In some cases, depending on the composition of the peptide (and hydrophobic sequences like yours favor this), the rate of interconversion is slow and one gets two peaks corresponding to the two different geometric forms. In effect, you are separating interconverting diastereomers. Whether or not you actually see the two peaks depends on whether or not the proline residue is a good binding site or is near a good binding site for the stationary phase. If there's a lysine residue nearby, then cation-exchange might be more sensitive to the interconversion than other modes, for example. Similarly, a standard C-18 column may or may not be able to separate them depending on the sequence, although the WP sequence seems promising in this regard. Such being the case, try any common combination for reversed-phase HPLC. If that doesn't work, then let us know what the sequence is of your peptide and we'll suggest alternative approaches.