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

• Simulations of both gradient and uniform mixed-mode LC columns are described. • Peak focusing is noted on stationary phase gradients under mobile phase gradients. • Orientation of stationary phase gradients effects peak width and retention time. • Experimental and simulation differences may suggest neighboring ligand effects. 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 C 18 -silica gradient stationary phase. Simulations show that peak widths for the solutes are dependent upon the fractional composition of C 18 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% C 18 where the column is oriented to have the C 18 dense region at the end. Experimentally, continuous stationary phase gradients were fabricated to have a total C 18 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% C 18 column. Simulations with a similar C 18 composition, however, predicted decreased retention times from the original C 18 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 C 18 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. [ABSTRACT FROM AUTHOR]