Investigation into reversed phase chromatography peptide separation systems part II: an evaluation of the robustness of a protocol for column characterisation

The robustness of the Peptide Reversed Phase Chromatography (RPC) Column Characterisation Protocol was evaluated using reduced factorial design, to ascertain the degree of control required for parameters including temperature, flow rate, dwell volume, a systematic shift in the gradient, amount of formic acid in the aqueous and organic, pH of the ammonium formate and amount of acetonitrile (%MeCN) in the strong solvent, where a loss of MeCN resulted in an unacceptable variation. Mitigations have been introduced to ensure the integrity of the data to allow RPC columns to be characterised using peptides as probes, with the definitive protocol described. In addition, the instrument and column batch to batch variability were assessed with good reproducibility.