Following previous studies of the use of non-porous monodisperse 1.5-pm noctyl-and n-octadecyl-bonded silicas in gradient elution of proteins, this work was aimed at elucidating further the properties of this novel column material for peptide and protein separations in comparison with wide-pore silicas.
First, it is demonstrated that with short columns (e.g., 35 x 8 mm I.D.) packed with these non-porous reversed-phase materials, mixtures of small peptides and mixtures of proteins can be very efficiently resolved. When the chain length of the bonded ligand was varied, the retention of a test set of proteins in gradient elution followed the ligand sequence C1 8 > Cs x C4 = p h enyl > C2 under constant elution conditions, and the selectivity remained unchanged. Comparison of the S values of these proteins, as determined from evaluation of the log k' vs. cp dependences with nonporous silicas and with a LiChrospher Si 1000 CB with identical accessible ligand surface areas per unit column volume, indicated lower values for the non-porous materials (k' = capacity factor; cp = molar fraction of organic solvent; S = slope of the plot of log k' vs. 40). The origin of this behaviour is discussed.