Improved resolution of glycoproteins by chromatography with concanavalin A immobilized on microparticulate silica via temperature-programmed elution.
The ability of the column temperature to control elution in the affinity chromatography of glycoproteins (e.g., ovalbumin and horseradish peroxidase) on silica immobilized concanavalin A has been studied. Column temperature programs can be achieved by placing a small HPLC column within a commercial mobile phase preheater assembly. It is shown that elution of adsorbed proteins can be initiated by changing the column temperature without altering the chemical composition of the mobile phase. Further, due to the enhancement in the rate of dissociation of the sample from the ligand, the peaks are narrowed. The resolution can be controlled by changing the initial temperature, dwell time at the initial temperature, and the rate of change of the temperature program. Addition of a competitive binding agent to the mobile phase decreases the temperature needed to elute strongly retained proteins. The effect of heating the column through many thermal cycles is assessed by periodically measuring the retention of a small monosaccharide that binds to the immobilized concanavalin A. The effect of two different immobilization procedures (glutaraldehyde and carbonyldiimidazole), as well as the effect of including a monosaccharide in the mobile phase, on the stability of the column is easily monitored by thermal elution chromatography. The effect of column temperature on the above glycoproteins has been assessed through studies of enzyme activities and anion exchange and isoelectric focusing patterns before and subsequent to temperature-programmed elution affinity chromatography.
Bergold
A F
AF
Carr
P W
PW
D002846:Chromatography, Affinity / Q000379:methods; D003208:Concanavalin A; D006023:Glycoproteins / Q000032:analysis*; D007202:Indicators and Reagents; D007525:Isoelectric Focusing; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D006023:Glycoproteins; D007202:Indicators and Reagents; D003208:Concanavalin A