Fundamental Understanding of Peptide Adsorption in Reversed-Phase Chromatography

Reversed phase chromatography is a relatively old technique that has been used extensively for the purification of small molecules. The stationary phases have thus been designed accordingly. Since peptides are charged molecules with a relatively large size (2-5kDa), they are often prevented to fully access all the stationary phase pores, leading thus to reduced column capacity. The effect of different factors (the peptide size and charges, the pore size distribution, the surface chemistry...) on the peptide pore accessibility and adsorption will be investigated and the impact on the separation performance will be clarified.

The effect of the peptide counter-ions on its retention behavior will be also addressed in this thesis. Peptides are usually produced by solid phase synthesis. At the end of the synthesis, they are cleaved from the surface and lyophilized in a TFA form (i.e. the peptide counter-ion is trifluoroacetate). However, TFA is toxic and has to be often replaced by an acetate counter-ion by the end of the purification. The exchange of TFA counter-ion by acetate is however very difficult due to the high affinity of TFA for the peptide. This is usually done during the chromatographic steps by replacement of the TFA counter-ion by the buffer counter-ion. This leads thus to very complex chromatographic system, where the peptide counter-ions change during the chromatographic runs, on an extent depending on the counter-ions affinity to the peptide. Since the counter-ions affect the peptide adsorption properties, unexpected behavior such as distorted peak shape and peak-splitting arises. A fundamental understanding of the counter-ion effect on the peptide retention behavior is thus required to improve the separation performances.

Contact Person: Rushd Khalaf