Comparative study of Zorbax Bio Series GF 250 and GF 450 and TSK-Gel 3000 SW and SWXL columns in size-exclusion chromatography of proteins.

Abstract A reduction of the mean particle diameter of silica-based packings in the size-exclusion chromatography (SEC) of proteins to about 5 μm generates the expected increase in column plate number over the traditional 10 μm SEC columns, as demonstrated for the Zorbax Bio Series GF 250 and GF 450 and TSK-Gel 3000 SWXL columns. The slightly lower column efficiency of the TSK-Gel 3000 SWXL compared with the GF 250 column is compensated by the fact that the phase ratio of the 3000 SWXL column is higher by a factor of two. Hence both columns show nearly the same peak capacity of about 20–30. When the ionic strength of the eluent was changed by varying the salt concentration, the elution volum…

BONDED SILICA PHASES FOR THE SEPARATION OF BIOPOLYMERS BY MEANS OF COLUMN LIQUID CHROMATOGRAPHY

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…

Affinity chromatography with triazine dyes immobilized onto activated non-porous monodisperse silicas

Abstract Non-porous monodisperse silicas with a particle diameter of 2.1 μm were modified with different silanes for immobilization of various triazine dyes including Procion Red HE3B, Procion Red MX5B, and Cibacron Blue F3GA. Lactate dehydrogenase and malate dehydrogenase from different species and aldehyde reductase from rat brain were purified by affinity elution using the substrate of the enzyme and NADH. With Cibacron F3GA the selectivity for NADH-dependent enzymes was higher than with the two Procion dyes. The utility of these immobilized triazine dye systems on non-porous silica supports for the rapid separation of Cohn Fraction III plasma proteins, including plasminogen, is also des…

Separation of plasma membrane proteins of cultured human fibroblasts by affinity chromatography on bonded microparticulate silicas.

Abstract Adsorbents for high-performance affinity chromatography were prepared by bonding proteins and reactive Procion triazine dyes to 3-isothiocyanatopropyl- and 3-aminopropylsilicas. The materials prepared were used successfully in the separation of hydrophobic plasma membrane proteins of cultured human fibroblasts. The data obtained show that the reaction of 3-isothiocyanatopropyltriethoxysilane (ITCPS) with the surface hydroxyl groups of silica yields a new and convenient route to preparing an “activated carrier” that is capable of coupling with potential affinity ligands containing amino functional groups. The reaction and bonding procedures of 3-isothiocyanatopropyltriethoxysilane a…