High Performance Liquid Chromatography of Small and Large Molecules with Nonporous Silica-Based Stationary Phases

Abstract

Nonporous monodispersed, microspherical silica particles, having mean particle diameters of 0.8 and 1.1 μm, were surfacel-modified with octadecyl or triphenyl functions as well as with lectin proteins for high performance liquid chromatography of small and large molecules. “Monomeric” or “polymeric” octadecyl-silica (C18-silica) bonded phases were formed, depending on the silane compounds used. Monomeric C18-silica stationary phases yielded high resolution in the separation of 2-pyridylamino derivatives of xyloglucan oligosaccharides and proteins. The effect of particle size of the support on resolution was also examined. For comparative study, the chromatographic behavior of large molecular weight proteins on a triphenyl-silica stationary phase was also conducted. Polymeric C18-silica stationary phases yielded higher phase ratios and better support surface coverage which proved useful in the separation of small hydrophilic species such as derivatized chitooligosaccharides of lower degree of polymerization (d.p.). The slopes of the linear dependence of logarithmic retention factor on volume percent of organic modifier were determined for the monomelic and polymeric C18-silica stationary phases with low molecular weight aromatic compounds. Lectin affinity stationary phases with lens culinaris agglutinin and wheat germ agglutinin covalently bound to nonporous silica support were examined for the applications of nonporous stationary phases in lectin affinity chromatography of glycoproteins. In all cases, rapid separations in the time scales of seconds and minutes could be obtained because of the absence of mass transfer resistance in the nonporous stationary phase matrices.