Characterisation of a new chromatography matrix for peptide molecular mass determination

The gel permeation behaviour of protein, small peptide and amino acid standards was assessed on a Superdex-Peptide column, using acetonitrile (30 or 70% containing 0.1% trifluoroacetic acid, TFA) as mobile phase. Efficient separation of the standards was observed using 30% acetonitrile; however, non-ideal gel permeation effects were observed for some charged amino acids and peptides containing hydrophobic residues. Retardation of the hydrophobic peptide standards was significantly reduced/eliminated using 70% acetonitrile as the mobile phase. However, chromatography with 70% acetonitrile was not suitable for molecular mass determination of proteins and free amino acids. The chromatographic behaviour of hydrolysates of β-lactoglobulin (β-lg) and whey protein concentrate on Superdex-Peptide and on TSK-2000SW columns was compared. Better separation of the hydrolysate peptides was obtained on the Superdex-Peptide column.