Retention characteristics of protonated mobile phases injected into deuterated mobile phases in capillary liquid chromatography (LC) using on-line nuclear magnetic resonance (NMR) detection

The behavior of protonated binary solvents injected into deuterated binary mobile phases in capillary LC is studied with NMR. Specifically, the solvent elution is followed on-flow with a capillary LC coupled to a 900 nL volume microcoil NMR probe. A range of identical composition 5% protonated (and 95% deuterated) solvents is injected into composition-matched deuterated mobile phases of CD₃CN/D₂O and CD₃OD/D₂O. The protonated components separate for all solvent combinations except at 80% CD₃CN/20% D₂O and ∼ 72% CD₃OD/28% D ₂O where only a single retention time is observed. The more hydrophilic protonated component, HOD, elutes first with higher percentages of hydrophilic solvent, D₂O, in the mobile phase whereas retention is reversed with the higher percentage of the more hydrophobic solvent (CD ₃CN and CD₃OD) in the mobile phase. The hydrophilic/hydrophobic nature of the chromatographic system as a function of mobile phase composition is characterized by following the retention times of protonated solvents.