L17 - Key Factors in Reverse Phase Method Development of Peptides

Peptides exist over a range of sizes and chemical properties. A variety of chromatographic techniques have been used to purified peptides. Reverse phase chromatography offers very high resolution, ideal to purify target peptides from close related impurities. Unfortunately, the principles of reverse phase do not apply in the same way as for smaller molecules. Secondary and tertiary structures on peptides create unpredictable behaviours. A broader range of parameters affect and to a higher extent the chromatography. The type of mobile phase, organic solvent used, and modifiers play a big role. Conditions of operation like gradient slopes, flow rate and temperature need to be optimised and controlled very tightly. Choice of column is very important too. Pore size, bonded phase, surface chemistry, column dimensions and particle size need to be carefully chosen depending on the application. Finally, column to column variability needs to be tightly controlled.

To provide an experimental framework for describing and modelling peptide separations, we have examined all these variables.

In this short presentation we review with the mean of some examples those key factors and set the basis for systematic optimization of peptide separations.