Peptide Purification by Reversed‐phase HPLC

Preparative reversed‐phase HPLC (RP‐HPLC) was carried out on a Luna C₁₈ column from Phenomenex (10‐µm particle size, 100Å pore size; 250 × 21.2 mm² I.D.). The column was equilibrated in 80% eluent A/20% eluent B, where eluent A is 0.2% aq. trifluoracetic acid (TFA) and eluent B is 0.15% TFA in CH₃CN. The sample (800 mg) was dissolved in 20% aq. CH₃CN containing 0.2% TFA to a concentration of 5 mg ml⁻¹, then loaded onto the column at a flow‐rate of 5 ml min⁻¹. The column was then eluted with 80% eluent A/20% eluent B for 30 min at 10 ml min⁻¹, followed by a shallow linear AB gradient of 0.1% B/min up to 50% CH₃CN. Fractions were collected every 2 min. Subsequent fraction analysis was carried out on a Luna C₁₈ RP‐HPLC column (5‐µm particle size, 100Å pore size; 250 × 3 mm I.D.) using a linear AB gradient (1% B/min) from starting conditions of 80% A/20% B. Purity of fractions was also verified by LC/MS using a HALO Peptide ES‐C18 2.0 µm, 2.1 mm I.D × 100 mm column from Advanced Materials Technology, USA. Pooled fractions containing purified material were subsequently lyophilized.