Following filtration through 0.45 µm Millipore filter the peptide solutions were loaded on to column via multiple 5 mL injections. Preparative C18 column (Gemini, 5 µm particle size, 100 Å pore size, 21.2 mm × 250 mm) from Phenomenex was used with a mixture of water and acetonitrile (both containing 0.1% (v/v) TFA) as eluent at a flow rate of 10 mL/min. The detection wavelength was 220 nm. Fractions containing the desired peptides were lyophilized.