Separation of tryptic peptides by HPLC. The crude radioactive peptide from 0.1% TFA/Acetonitrile solvent system was reapplied to Vydac C₁₈ HPLC column equilibrated with 20 mmol/L ammonium acetate, pH 6.1. The peptide peaks were eluted at 1 mL/min with a linear gradient of 20 mmol/L ammonium acetate in 50% acetonitrile after a 10 minutes wash. The only radioactive peak, indicated by an arrow, was pooled and sequenced.