Experimental- and simulation-based investigations of coupling a mobile phase gradient with a continuous stationary phase gradient

Abstract

This work seeks to explore and understand the effects of column orientation and degree of modification of continuous stationary phase gradient columns under a mobile phase gradient using both simulations and experiments. Peak parameters such as retention times, peak widths and resolution are obtained for five phenolic compounds on a C18-silica gradient stationary phase. Simulations show that peak widths for the solutes are dependent upon the fractional composition of C18 and orientation of the stationary phase gradient when coupled to a mobile phase gradient. Also, when compared to a simulated uniform mixed-mode column, peak widths reach a minimum on the gradient column with a coverage higher than 50% C18 where the column is oriented to have the C18 dense region at the end. Experimentally, continuous stationary phase gradients were fabricated to have a total C18 composition of 78% of the original uniform column with an exponential profile using a previously described destructive controlled rate infusion method. Under gradient mobile phase conditions, experimental retention times for the gradient column showed a significant increase compared to the original 100% C18 column. Simulations with a similar C18 composition, however, predicted decreased retention times from the original C18 column. A statistical increase in the retention time of protocatechuic acid and decrease in the peak width of tyrosol, caffeic acid, and coumaric acid were noted when the gradient column was oriented to have the C18 dense region located near the detector. Collectively, combining gradients in both the mobile and stationary phases can yield interesting neighboring ligand effects and peak broadening/focusing effects.