Destructive stationary phase gradients for reversed-phase/hydrophilic interaction liquid chromatography

The use of stationary phase gradients for liquid chromatography (LC) is a promising new strategy to allow for specific control over the selectivity of a separation by having a gradual change in the ligand density along the length of the column. Unfortunately, there have been very few, if any, methods to prepare continuous stationary phase gradients on traditional packed LC columns. In this work, destructive methodologies are used to create stationary phase gradients on commercial C₁₈ columns by infusing trifluoroacetic acid (TFA) onto the column through controlled rate of infusion (CRI). The introduction of TFA via CRI while the column is heated at 80 °C promotes acid hydrolysis of the alkylsilane ligand in a gradient fashion. Characterization with scanning electron microscopy and Barrett-Joyner-Halenda pore size distributions of the stationary phase after fabrication of the destructive gradient establishes that the chromatographic support was not damaged during the procedure. The shape of the gradient was examined using thermogravimetric analysis (TGA) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. TGA and ATR-FTIR showed an increase in the percent carbon loss along the length of the column, indicating that there was an increase in the C₁₈ ligand from the front to the end of the column. Two selectivity tests demonstrated a decrease in the hydrophobicity and increase in the silanol activity of the stationary phase gradient from the uniform C₁₈ counterpart. Additionally, the fabrication of the destructive stationary phase gradient resulted in two different surface functionalities allowing hydrophobic and hydrophilic interactions with analyte species depending on the mobile phase composition. Plots of the log of retention factor versus percent acetonitrile illustrated that these stationary phase gradients have two separation mechanisms: reversed-phase (RP) and hydrophilic interaction. Coupling the stationary phase gradient with a mobile phase gradient shows differences in the peak widths and the resolution of phenolic compounds, indicating that the orientation of the stationary phase gradient has the potential to enhance the resolution of a separation. With this methodology, stationary phase gradients can be fabricated on previously used RP columns, allowing for these columns to be repurposed.