Chaotropic Effects in Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at Preparative Scale

Chromatographic separation, analysis and characterization of complex highly polar analyte mixtures can often be very challenging using conventional separation approaches. Analysis and purification of hydrophilic compounds have been dominated by liquid chromatography (LC) and ion-exchange chromatography (IC), with supercritical fluid chromatography (SFC) moving towards these new applications, beyond traditional chiral separations. However, the low polarity of supercritical carbon dioxide (CO2) has limited the use of SFC for separation and purification in the bioanalytical space, especially at preparative scale. Reaction mixtures of highly polar species are strongly retained even using polar additives in alcohol modifier/CO2 based eluents. Herein we overcome these problems by introducing chaotropic effects in SFC separations using a non-traditional mobile phase mixture consisting of ammonium hydroxide combined with high water concentration in alcohol modifier and carbon dioxide. The separation mechanism was...