High-Resolution Reversed-Phase Liquid Chromatography of Peptide Separations
The development of a good and reproducible separation of peptides with small differences in the amino acid composition is difficult and challenging. In this paper, we present a strategy for the rapid development of rugged and validatable peptide maps using reversed-phase gradient HPLC. The impact of the reversed-phase column characteristics (pore size, pore volume, hydrophobicity, hydrolytic stability), the mobile phase modifier, the temperature, the type of acid and its concentration will be discussed. Also, both the column and batch-to-batch reproducibility of the packing material will be emphasized.