1. Principles of Reversed-Phase Chromatography
Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) relies on a non-polar stationary phase (typically silica particles coated with octadecyl carbon chains, or C18) combined with a polar mobile phase consisting of water and acetonitrile modified with an ion-pairing agent such as 0.1% Trifluoroacetic Acid (TFA).
2. Resolution of Truncated and Deletion Sequences
During solid-phase peptide synthesis (SPPS), incomplete coupling or deprotection reactions can generate premature sequence truncations. Because even single amino acid deletions significantly alter the hydrophobicity profile of the peptide, high-resolution C18 columns are capable of resolving these contaminants from the desired full-length target.
3. Interpreting Chromatographic Peak Area
Purity is determined by integrating the ultraviolet absorption area (typically measured at 214 nm or 220 nm, corresponding to the peptide backbone amide bond absorption) of the primary target peak relative to the sum total of all eluted impurities.