Reversed-Phase HPLC
The most commonly used HPLC variant for peptide analysis, separating peptides based on hydrophobicity using a non-polar stationary phase and polar mobile phase. Reversed-phase HPLC provides high resolution for distinguishing the target peptide from closely related impurities and degradation products.
Technical Context
RP-HPLC uses a non-polar stationary phase (C18, C8, C4 bonded silica — C18 is most common for peptides <5 kDa, C4 for larger peptides/proteins where C18 binding may be too strong) and a polar mobile phase. Peptides bind to the non-polar stationary phase via hydrophobic interactions; increasing organic solvent (acetonitrile) concentration during gradient elution disrupts these interactions, eluting peptides in order of increasing hydrophobicity. For peptide purity analysis: the area percentage of the target peptide peak relative to total peak area gives the purity value. Related impurities (deletion peptides, truncated sequences, oxidised forms) typically elute close to the target peak, requiring sufficient resolution. Ion-pair reagents (TFA, formic acid) protonate basic residues and pair with acidic residues, improving peak shape and resolution. Peptide retention time is predictable from amino acid composition using hydrophobicity scales.