ABSTRACT
Stationary phase gradients on monolithic silica columns have been successfully and reproducibly prepared and characterized with comparisons made to uniformly modified stationary phases. Stationary phase gradients hold great potential for use in liquid chromatography (LC), both in terms of simplifying analysis as well as providing novel selectivity. In this work, we demonstrate the creation of a continuous stationary phase gradient on in-house synthesized monolithic columns by infusing an aminoalkoxysilane solution through the silica monoliths via controlled rate infusion. The presence of amine and its distribution along the length of gradient and uniformly modified columns were assessed via X-ray photoelectron spectroscopy (XPS). XPS showed a clear gradient in surface coverage along the length of the column for the gradient stationary phases while a near uniform distribution on the uniformly modified stationary phases. To demonstrate the application of these gradient stationary phases, the separations of both nucleobases and weak acids/weak bases on these gradient stationary phases have been compared to uniformly modified and unmodified silica columns. Of particular note, the retention characteristics of 11 gradient columns, 5 uniformly modified columns, and 5 unmodified columns have been tested to establish the reproducibility of the synthetic procedures. Standard deviations of the retention factors were in the range from 0.06 to 0.5, depending on the analyte species. We show that selectivity is achieved with the stationary phase gradients that are significantly different from either uniformly modified amine or unmodified columns. These results indicate the significant promise of this strategy for creating novel stationary phases for LC.
ABSTRACT
Continuous multi-component gradients in amine and phenyl groups were fabricated using controlled rate infusion (CRI). Solutions prepared from either 3-aminopropyltriethoxysilane (APTEOS) or phenyltrimethoxysilane (PTMOS) were infused, in a sequential fashion, at a controlled rate into an empty graduated cylinder housing a vertically aligned thin layer chromatography (TLC) plate. The hydrolyzed precursors reacted with an abundance of silanol (SiOH) groups on the TLC plates, covalently attaching the functionalized silane to its surface. The extent of modification by phenyl and amine was determined by the kinetics of each reaction and the exposure time at each point along the TLC plate. The local concentrations of phenyl and amine were measured using diffuse reflectance spectroscopy and X-ray photoelectron spectroscopy, respectively. The profile of the multi-component gradients strongly depended on the order of infusion, the direction of the gradient and the presence of available surface silanol groups. A slightly higher amount of phenyl can be deposited on the TLC plate by first modifying its surface with amine groups as they serve as a catalyst, enhancing condensation. Separation of water- and fat-soluble vitamins and the control of retention factors were demonstrated on the multi-component gradient TLC plates. Uniformly modified and single-component TLC plates gave different separations compared to the multi-component gradient plates. The retention factors of the individual vitamins depended on the order of surface modification, the spotting end, and whether the multi-component gradients align or oppose each other.
