High speed gradient elution reversed phase liquid chromatography of bases in buffered eluents

Abstract

In this work we determined when the state of thermodynamic (full) equilibrium, i.e. time-invariate solute retention, was achieved in gradient elution reversed-phase chromatography. We investigated the effects of flow rate, temperature, organic modifier, buffer type/concentration, stationary phase type, n-butanol as eluent additive, and pore size. We also measured how selectivity varied with reequilibration time. Stationary phase wetting and the ability of the stationary phase to resist changes in pH strongly affect the time needed to reach full equilibrium. For example, full equilibrium is realized with many endcapped stationary phases after flushing with only two column volumes of acetonitrile-water containing 1 % (v/v) n-butanol and 0.1 % (v/ v) trifluoroacetic acid. Trends in retention time (< 0.010 min) and selectivity become quite small after only five column volumes of reequilibration. We give practical guidelines that provide fast full equilibrium for basic compounds when chromatographed in buffered eluents.

An experimental study of sampling time effects on the resolving power of on-line two-dimensional high performance liquid chromatography

“…In addition, the small dimensions of this column also allow us to have a good detection sensitivity on the second dimension. Finally, the small dimensions of this column together with the high flow rate ensures a short system dwell time and fast column re-equilibration [42,43]. Carbon-clad zirconia was chosen as the second dimension phase for its good chemical and mechanical stability at high temperature, high flow rate conditions, and most importantly its high degree of orthogonal selectivity relative to other RPC phases [4].…”

Adsorption and recovery issues of recombinant monoclonal antibodies in reversed-phase liquid chromatography†

“…Two solutions were proposed to reduce the specific adsorption phenomenon of mAbs, including the use of high mobile‐phase temperature (up to 80–90°C) and/or the addition of ancillary solvents (e.g., 1–3% n ‐butanol) to the mobile phase . This last strategy was previously described by Scott and Simpson in the 1980s and recently updated to reduce the time required for column equilibrium between successive RPLC gradients. Scott and Simpson first described the strong adsorption of n ‐butanol at the surface of the stationary phase .…”

“…Scott and Simpson first described the strong adsorption of n ‐butanol at the surface of the stationary phase . According to Carr and coworkers, the hydroxyl group of n ‐butanol strongly interacts either with the surface silanol groups or with water adsorbed on the silanol groups to “shield” the acidic silanols .…”

Theoretical and practical interest in UHPLC technology for 2D-LC

“…However, it was shown that only two column volumes (x = 2) can provide good runto-run repeatability in UHPLC conditions, even with charged compounds [23][24][25]. Starting from these values, the requirement in on-line LC x LC becomes:…”