Separation of hydrophobic peptide polymers by size-exclusion and reversed-phase high-performance liquid chromatography

Abstract

Four synthetic leucine polymers of 13,22,26 and 30 residues with the sequence Ac-(Leu) 10-(Lys) 2-Ala-amide and (Lys) 2-Gly-(Leu) n -(Lys) 2-Ala-amide, where n = 16, 20 and 24, were used to determine the utility of reversed-phase chromatography in separating extremely hydrophobic peptides. Two mobile phases were examined, water—acetonitrile and water—2-propanol, both containing 0.1% trifluoroacetic acid (TFA). 2-Propanol was the preferred organic solvent, and the hydrophobic peptides (13, 22 and 26 residues) were resolved on the Altex Ultrapore C 3 column (300-Å pore size, 5-μm particle size, 2.9% carbon loading) in the 25–50% range of a linear AB gradient (A = 0.1% aq. TFA; B = 0.1% TFA in 2-propanol) increasing at 1% B/min. This procedure permits detection by absorbance at 210 nm, and the peptides can be recovered by evaporation or lyophilization. The 30 residue leucine polymer could not be eluted from the reversed-phase column. These peptides showed non-ideal behavior on size-exclusion chromatography in 0.1% aq. TFA containing 50% acetonitrile. However, this caused no problem in peptide purification. The resolution on a TSK G3000SW column was superior to that obtained on TSK G2000SW.