Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography. III. Retention and selectivity of proteins and peptides in gradient elution on non-porous monodisperse 1.5-microns reversed-phase silicas.

Following previous studies of the use of non-porous monodisperse 1.5-microns n-octyl- 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 C18 greater than C8 approximately C4 approximately phenyl greater than 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. phi dependences with non-porous silicas and with a LiChrospher Si 1000 C8 with identical accessible ligand surface areas per unit column volume, indicated lower values for the non-porous materials (k' = capacity factor; phi = molar fraction of organic solvent; S = slope of the plot of log k' vs. phi). The origin of this behaviour is discussed.