High-performance liquid chromatography of peptides on a microspherical carbon column

The properties of a microspherical carbon column for the separation of peptides and proteins are described. One hundred and thirty-three peptides, with from 1 to 148 amino acid residues, were applied on a carbon column and eluted with a linear gradient of acetonitrile [10-70% (v/v), 30 min] in 0.1% (v/v) trifluoroacetic acid solution. The elution behaviour of the peptides on the carbon column and on an octadecylsilica (ODS) column were compared under the same elution conditions. Relationships between the logarithm of the hydrophobicity of peptides and their capacity factors were approximately linear on both columns, but some exceptions were observed with peptides with aromatic residues, indicating stronger adsorption on the carbon than on the ODS column. These results suggest that the major factor for the separation of peptides on both the carbon and ODS columns is hydrophobic interactions. The stronger adsorption of aromatic side-chains in peptides on the carbon column could be understood by an interaction based on the aromatic or graphitic nature of the surface of the microspherical carbon packings.