Improvement of Analysis and Transferability in Peptide Purification: From HPLC to FPLC and Back Again

Optimal purification, high purity and robust purity control for synthetic peptides are critical, as even minor impurities can alter biological activity, distort analytical results and compromise downstream applications. Reliable translation of analytical HPLC results to preparative FPLC conditions remains challenging due to system-specific differences in column geometry, gradient formation and mobile-phase chemistry. This study addresses these limitations through a systematic evaluation of chromatographic parameters influencing peptide selectivity, resolution and transferability. Using a defined peptide impurity library, the effects of gradient steepness, flow rate, temperature and mobile-phase modifier were quantified, revealing that flow-rate optimization and modifier choice have the greatest impact on separation quality. A correction equation was developed to compensate for system-dependent deviations, reducing transfer errors in elution percentage from approximately 17% to less than 5%. The optimized workflow enabled initial preparative purifications with purities above 90% and yields exceeding 30%. Additionally, substitution of trifluoroacetic acid with formic acid was explored as a greener modifier, providing selective improvements in separation performance. The approach establishes a practical and sustainable workflow for the transfer of HPLC-to-FPLC methods for peptide purification.