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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 54-61 |
| Number of pages | 8 |
| Journal | Journal of Chromatography A |
| Volume | 1192 |
| Issue number | 1 |
| DOIs | |
| State | Published - May 23 2008 |
Bibliographical note
Funding Information:The authors acknowledge financial support from the National Institutes of Health (grant # 5R01GM054585-09). We also thank Professor Peter Schoenmakers for his critical comments and suggestions involving the importance of stationary phase wetting on full equilibrium. We also thank Dr. Lloyd R. Snyder for many helpful discussions during the course of the work.
Keywords
- Basic drugs
- Equilibration
- Flush-out volume
- Gradient elution
- Speed