Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography

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 approxima…

ChemInform Abstract: Nonporous Silica Microspheres in the Micron and Submicron Size Range: Manufacture, Characterization and Application

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography

Abstract The reversed-phase chromatography of proteins by gradient elution with acidic, low-ionic-strength aqueous—organic eluents is often associated with losses of the biological activity of the protein. In this study, the enzymatic activities of catalase, horseradish peroxidase and pepsin were examined under static and dynamic column conditions on non-porous, monodisperse 1.5-μm reversed-phase silicas with various n -alkyl ligands. Catalase readily lost its enzymatic activity under the influence of the acidic aqueous—organic eluents in the absence of the reversed-phase packing, whereas peroxidase was partially deactivated as a result of combined mobile phase and stationary phase effects …

Packing technology, column bed structure and chromatographic performance of 1-2-μm non-porous silicas in high-performance liquid chromatography

This work is aimed at further elucidating the aggregation behaviour of micron- and submicron-size non-porous silicas and the column performance of 1–2-μm C18 silicas in reversed-phase high-performance liquid chromatography of low-molecular weight compounds. It is demonstrated that highly ordered, dense, porous aggregates of such silica beads were obtained by gravity settling and centrifugation. The slurry techniques applied at constant flow-rate and a pressure up to 50 MPa provided less-ordered aggregates, but generated an acceptable performance of columns when 1–2-μm C18 silica beads were employed. To operate columns of 53 mm × 4.6 mm I.D., the maximum flow-rate needs to be ca. 2.5 ml/min …

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography

Abstract In the separation of proteins and peptides by the various modes of high-performance liquid chromatography, the nature of the substrates requires the use of microparticulate silica packings with bonded ligands of appropriate design. Agglomeration of monodisperse silica hydrosols of defined size distribution into beaded particles provides a useful method of controlling the pore size, the size distribution, the particle porosity and surface area of these packings. The particle porosity is shown to be a major factor governing the packing density and packing stability of the column. For size-exclusion chromatography of proteins, parent silicas of pore size 10 and 100 nm with a narrow po…

Hysteresis in nitrogen sorption and mercury porosimetry on mesoporous model adsorbents made of aggregated monodisperse silica spheres

Abstract An elucidation of the hysteresis phenomena in nitrogen sorption (NS) and mercury porosimetry (MP) on porous aggregates of non-porous silica spheres of particle diameter 90–1000 nm is presented. First, it is demonstrated that highly ordered and dense aggregates of a porosity of 26–30% are formed by gravity settling and centrifugation. A hexagonal and cubic close-packed structure within the layers and a vertical stacking between the layers were observed. Minor deteriorations were seen between the ordered domains. Second, the pore size distribution (PSD) curves calculated from the adsorption branch of the nitrogen isotherms by means of the Cohan equation and from the desorption branch…

Evaluation of advanced silica packings for the separation of biopolymers by high-perforamnce liquid chromatography

Non-porous monodisperse 1.5-μm silicas were allowed to react with (A) and (B) N-acetylaminopropyltriethoxysilane to generate bonded phases useful in high-performance hydrophobic-interaction chromatography (HIC). Differences in the selectivity were observed between he amide and the ether phase. Peak capacities between 10 and 30 were achieved for several proteins with the amide and ether phase packed into columns of 36 × 8 mm I.D. and elution of the proteins under chromatographic conditions in which the gradient volume, VG, was held constant by varying the gradient time between 20 and 2.5 min and the flow-rate between 0.5 and 4.0 ml/min. The S values derived from the dependences of log k′ on …