In situ silanization for continuous stationary phase gradients on particle packed LC columns

Mobile phase gradients are ubiquitous in liquid chromatography (LC); stationary phase gradients are not. Variation in the ligand density along the length of column can provide an important means to optimize separations. However, a major impediment relates to the development of methodologies to fabricate continuous gradient stationary phases, particularly on particle packed columns. For the first time, we demonstrate an in situ silanization approach based on the controlled rate infusion method to create a stationary phase gradient. This was accomplished by infusing a phenylbutyltrimethoxysilane solution through an in-house particle packed column for 1 h, with one end of the column being exposed longer than the other. Raman spectroscopy reveals a steep gradient over the first centimeter of the column followed by a plateau, indicating successful in situ modification. N2 sorption experiments confirm the organosilane does not block the pores of the silica. The reversed phase (RP) and hydrophilic interaction liquid chromatography (HILIC) nature of the gradient stationary phase were evaluated using hydrophobic and hydrophilic probe analytes. The stability of the retention factors (RSD < 6%) and the column-to-column reproducibility (4% < RSD < 9%) were satisfactory. Surprising differences were observed in the amount of modification and the peak shapes for the gradient versus uniform stationary phases, which are partially attributed the nature of the on-column modification procedure. This work serves as a proof-of-concept for the constructive fabrication of continuous stationary phase gradients on particle packed columns and will hopefully stimulate future developments.