Fabrication and Characterization of a Reversed-Phase/Strong Cation Exchange Stationary Phase Gradient.

Continuous stationary phase gradients for liquid chromatography (LC) have been recently shown to be a promising method of altering selectivity. In this work, we present the first multicomponent continuous stationary phase gradient for separations involving both reversed-phase (RP) and strong cation exchange (SCX) mechanisms. These columns are fabricated using a two-step methodology based on controlled rate infusion (CRI). First, destructive CRI creates a gradient of excess silanol groups along a uniform C18 column. Next, these columns are infused with 3-mercaptopropyltrimethoxysilane (MPTMOS), which bonds to the excess silanol groups. The terminal thiols of the MPTMOS ligands are oxidized with H2O2 to create the sulfonate functionality (SO3-) needed for SCX separations. The success of the modification procedure is characterized by thermogravimetric analysis and X-ray photoelectron spectroscopy. The stability of the C18-SO3- gradients were found to have less than 5 % retention loss and the column-to-column reproducibility had a relative standard deviation under 9 %. The peak asymmetry factors for seven biogenic amines were found to be between 1.03 ± 0.04 to 1.30 ± 0.02, which illustrates minimal peak tailing due to poor packing and residual silanol groups. Characterization of the gradient columns using an isocratic mobile phase showed a unique elution order compared to a uniform C18 and SO3- columns. At lower counterion concentrations, more than 48 % of the overall retention on the gradient stationary phase is due to a SCX mechanism. Meanwhile, the RP mechanism was shown to predominate at higher counterion concentrations on the gradient columns (SCX retention contribution less than 40 %). Coupling the stationary phase gradient to a salt gradient in the mobile phase showed that the gradient phase provides a unique, intermediate selectivity to the uniform C18 and SO3- columns. Under an ACN mobile phase gradient, a significant increase (p < 0.003) in the retention times of three biogenic amines (15 - 16 %) was observed when the multicomponent gradient was oriented to have a high SO3- ligand density near the detector. This work serves as a proof-of-concept design for a multicomponent stationary phase gradient to continue fundamental studies into retention and encourage novel applications.

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

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.

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.

Vapor-Phase Plotting of Organosilane Chemical Gradients.

Vapor-phase plotting of organosilane-based self-assembled monolayer (SAM) gradients is demonstrated for the first time. Patterned SAMs are formed by delivering gas-phase organotrichlorosilane precursors to a reactive silica surface using a heated glass capillary. The capillary is attached via a short flexible tube to a reservoir containing the precursor dissolved in toluene. The proximal end of the capillary is positioned at an experimentally optimized distance of 30 µm above the substrate during film deposition. The capillary is mounted to a stepper-motor-driven X, Y plotter for raster scanning above the surface. Two different organotrichlorosilane precursors are employed in this initial demonstration: n-octyltrichlorosilane and 3-cyanopropyltrichlorosilane. The dependence of SAM deposition on ambient relative humidity, capillary-substrate separation, raster-scanning speed, and solvent viscosity and volatility is explored and optimum deposition conditions are identified. The optimized procedures are used to plot uniformly modified square "pads" and gradients of the silanes. Film formation is verified and the gradient profiles are obtained by sessile drop water contact angle measurements, spectroscopic ellipsometry measurements of film thickness, and X-ray photoelectron spectroscopy mapping. The resolution of the plotting process is currently in the millimeter range and depends on capillary diameter and distance from the substrate surface. Vapor-phase plotting affords a unique direct-write method for producing patterned and chemically graded SAMS that may find applications in microfluidic devices, planar chromatography, and optical and electronic devices.

Integrated Electrodes and Electrospray Emitter for Polymer Microfluidic Nanospray-MS Interface.

Interfacing of microfluidic devices to mass spectrometry has challenges including dilution from sheath liquid junctions, fragile electrodes, and excessive dead volumes which prevent optimum performance and common use. The goal of this work is to develop a stable nanospray chip-MS interface that contains easily integrated electrodes and an embedded capillary emitter to mitigate current chip-MS problems. This system uses a hybrid polystyrene-poly(dimethylsiloxane) (PS-PDMS) microfluidic platform with an embedded electrode and integrated capillary emitter used as the nanospray interface. Two chip designs were used to evaluate the performance, illustrate on-chip reaction capabilities. By direct infusion, this system showed good performance with LODs of GSH and caffeine of 9 nM and 1 nM, R2 of 0.996 and 0.992 and sensitivity of 12 counts/nM and 332 counts/nM over a linear dynamic range of 40 nM to 50 µM and 1 to 50 µM respectively. A reaction was performed on the chip with syringe pumps showing the oxidation of glutathione (GSH) to oxidized glutathione (GSSG) using H2O2. The on-chip reaction of GSH oxidation to GSSG, with online-MS detection, successfully demonstrate the stability and robustness of the nanospray interface.