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.