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

Abstract

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 sub/supercritical fluid chromatography (SFC) moving toward 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 the 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 nontraditional mobile phase mixture consisting of ammonium hydroxide combined with high water concentration in the alcohol modifier and carbon dioxide. The separation mechanism was here elucidated based on extensive IC-CD (IC couple to conductivity detection) analysis of cyclic peptides subjected to the SFC conditions, indicating the in situ formation of a bicarbonate counterion (HCO3 –). In contrast to other salts, HCO3 – was found to play a crucial role acting as a chaotropic agent that disrupts undesired H-bonding interactions, which was demonstrated by size-exclusion chromatography coupled with differential hydrogen–deuterium exchange-mass spectrometry experiments (SEC-HDX-MS). In addition, the use of NH4OH in water-rich MeOH modifiers was compared to other commonly used basic additives (diethylamine, triethylamine, and isobutylamine) showing unmatched chromatographic and MS detection performance in terms of peak shape, retention, selectivity, and ionization as well as a completely different selectivity and retention behavior. Moreover, relative to ammonium formate and ammonium acetate in water-rich methanol modifier, the ammonium hydroxide in water additive showed better chromatographic performance with enhanced sensitivity. Further optimization of NH4OH and H2O levels in conjunction with MeOH/CO2 served to furnish a generic modifier (0.2% NH4OH, 5% H2O in MeOH) that enables the widespread transition of SFC to domains that were previously considered out of its scope. This approach is extensively applied to the separation, analysis, and purification of multicomponent reaction mixtures of closely related polar pharmaceuticals using readily available SFC instrumentation. The examples described here cover a broad spectrum of bioanalytical and pharmaceutical applications including analytical and preparative chromatography of organohalogenated species, nucleobases, nucleosides, nucleotides, sulfonamides, and cyclic peptides among other highly polar species.

Supercritical fluid chromatography with post‐column addition of supporting electrolyte solution for electrochemical determination of tocopherol and tocotrienol isomers

“…In contrast to our approach to maintaining the conductivity of the electrochemical cell in SFC, it is expected that the water additive in a modifier, to which attention has recently been paid [40–42], is one of the solutions, because the water additive may improve the solubility of the electrolytes in the modifier. However, the content of water in the modifier (generally around 5 v/v%) appears to be not enough to drastically improve the solubilities of the electrolyte in the modifier based on a previous study for the solubility of the electrolyte in a mixed solvent [43].…”

A review of retention mechanism studies for packed column supercritical fluid chromatography

“…Glenne et al showed that the fraction of amines highly affected column efficiency and peak shape of basic pharmaceuticals used in the study under SFC conditions 69 . In 2019, Liu et al reported the effect of bicarbonate as a chaotropic agent that disrupted undesired H‐bonding interactions in separations of cyclic peptides using SFC when ammonium was used in water‐rich MeOH modifiers 70 . It was found that compared to traditional basic additives, using ammonium hydroxide in water‐rich MeOH modifiers resulted in a better performance in terms of the peak shape, analyte retention, selectivity, and ionization efficiency.…”

Emerging Separation Techniques in Supercritical Fluid Chromatography

“…In general, measurement time tends to be longer when multiple analytical columns are connected to each other. As the measurement time of SFC is generally shorter than HPLC, SFC × SFC 63) and HPLC × SFC 64,65) were developed for the achiral/chiral separation of pharmaceutics. Because of the complexity of a two-dimensional chromatography system that consists of multiple pumps, switch valves, and interfaces for different mobile phases, the introduction of SFC was challenging but succeeded as an established system.…”