Optimization of the recovery yield and of the production rate in overloaded gradient-elution reversed-phase chromatography.

To investigate the effects of various parameters on the production rate and on the recovery yield in overloaded reversed-phase gradient-elution chromatography, band profiles of binary mixtures of phenol and o-cresol were calculated using the experimental parameters of the distribution isotherms determined by binary frontal analysis. The effects of the feed volume and of the steepness of a continuous gradient (gradient time) of methanol in aqueous-organic mobile phases on the separation were studied. If the sample feed in a solvent with weak elution strength is used, combined effects of on-column enrichment, frontal chromatography and sharpening of the later eluted bands may enhance the production rate and the recovery yield. Steep continuous gradients offer better results than isocratic elution if the feed is dissolved in water and injected into the mobile phase with a higher elution strength, because larger volumes of dilute feed can be separated with high recovery yields.