We studied the run-to-run repeatability of the retention times of both non-ionizable and basic compounds chromatographed using buffered eluents. The effect of flow rate, organic modifier and other additives, buffer type/concentration, stationary phase type, batch-to-batch preparation of the initial eluent, gradient time, sample type and intra-day changes on retention repeatability were examined. We also assessed the effect of column storage solvent conditions on the inter-day repeatability. Although retention repeatability is strongly influenced by many parameters (flow rate, solvent compressibility compensation, precision of temperature control, and buffer/stationary phase type), our primary finding is that with a reasonable size column (15 cm × 4.6 mm (i.d.)) two column volumes of re-equilibration with initial eluent suffices to provide acceptable repeatability (no worse than 0.004 min) for both non-ionizable and basic analytes under a wide variety of conditions. Under ideal conditions (e.g. the right buffer, flow rate, etc.) it is possible to obtain truly extraordinary repeatability often as good as 0.0004 min. These absolute fluctuations in retention translate to worst case changes in resolution of 0.2 units and average changes of only 0.02 units.
Bibliographical noteFunding Information:
The authors acknowledge financial support from the National Institutes of Health (grant # 5R01GM054585-09). We also thank Agilent technologies for their generous donation of the SB-C 18 and XDB-C 18 columns used in this work.
- Basic compounds
- Gradient elution