Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

Abstract

The potential of enhanced-fluidity liquid chromatography, a subcritical chromatography technique, in mixed-mode hydrophilic interaction/strong cation-exchange separations is explored, using amino acids as analytes. The enhanced-fluidity liquid mobile phases were prepared by adding liquefied CO to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO to methanol/water mixtures resulted in increased retention of the more polar amino acids. The "optimized" chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced-fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced-fluidity mobile phase separation was governed by a mixed-mode retention mechanism of hydrophilic interaction/strong cation-exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.

Enhanced-Fluidity Liquid Chromatography–Mass Spectrometry for Intact Protein Separation and Characterization

“…The retention mechanism for columns under these conditions has been explained as a HIC-RPC mixed-mode interaction with some organic solvent added to the salt gradient to promote protein elution in a reasonable time frame. This mixed-mode feature is desirable for our investigation of the EFLC-MS system for proteins because, in order to add liquefied CO 2 to a HIC salt gradient, an organic cosolvent (such as methanol, ethanol, and acetonitrile) is required to avoid solvent demixing …”

Two‐dimensional separation techniques using supercritical fluid chromatography

“…While most iterations of 2D‐SFC have used RPLC and NPLC 1 D columns, the use of 1 D IEX and SEC separations coupled to 2 D SFC columns is inevitably on the horizon. Although the use of ion‐exchange columns and ion‐pairing reagents has been demonstrated with SFC for the separation of ionic compounds [87–90], the use of hydrophobic, high proton affinity tags to derivatize polar analytes and membrane proteins offers unique opportunities to leverage multiple aspects of analyte preconcentration in IEX with high‐speed SFC separations in a combined 2D method. Additionally, polymer analysis was undertaken with SFC × SFC [78], but the use of SEC may offer a more orthogonal approach for improved resolution of complex polymers based on previous SEC‐capillary SFC methods [57,58].…”

Zero electrical power pump for portable high-performance liquid chromatography

“…Techniques such as enhanced-fluidity LC involves the addition of liquefied CO 2 to conventional liquid mobile phases and has been shown to provide enhanced diffusivity, faster solute mass transfer, and reduced system backpressure. 12,13 We have not, as yet, explored how this effect might be exploited using our system. We have found the low solubility of nitrogen permits the driving of mobile phase without necessitating a physical barrier or membrane to isolate the phases.…”