INTRODUCTIONTwo types of columns are commonly used for the separation of peptides by HPLC. A single-phase column contains the reverse-phase resin C18, which interacts with the hydrophobic moieties of the peptides. Peptides resulting from digestion of simple mixtures of proteins are loaded onto the single-phase column and eluted into the mass analyzer using an increasing gradient of an organic solvent. Peptides resulting from the digestion of more complex mixtures of proteins are resolved using a biphasic column. This column integrates both a strong cation exchange SCX resin, which interacts with peptides as a result of their positive charge, and a reverse-phase C18 resin, packed in tandem. Peptides initially interact with the SCX resin and are eluted into the C18 resin by ammonium acetate that competes for the peptide-binding sites. Peptides are then eluted from the C18 resin into the mass analyzer. This process is repeated using increasing concentrations of ammonium acetate to differentially elute peptides in a stepwise fashion. The biphasic column also uses an additional C18 reverse-phase resin linked through an Inline MicroFilter Assembly (Upchurch) to desalt the peptides prior to loading onto the SCX. The desalting and biphasic columns are combined to give an integrated desalting/biphasic column.