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

Abstract

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 by 4.6 mm (i.d.)) two column volumes of reequilibration 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 minutes. These absolute fluctuations in retention translate to worst case changes in resolution of 0.2 units and average changes of only 0.02 units.

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:…”

Instrument parameters controlling retention precision in gradient elution reversed-phase liquid chromatography

“…Additionally precise measurement of retention times allows the prediction of separations by using gradient elution training data through methods described by Snyder [19]. There have been a number of studies of the reproducibility of retention time in liquid chromatography that emphasize the importance of column equilibration [12, 20-32]. It is possible to achieve run to run gradient elution retention reproducibility to ± 0.002 min (standard deviation) in a bit more than two column volumes of re-equilibration (so-called “repeatable equilibrium”) [20].…”

“…There have been a number of studies of the reproducibility of retention time in liquid chromatography that emphasize the importance of column equilibration [12, 20-32]. It is possible to achieve run to run gradient elution retention reproducibility to ± 0.002 min (standard deviation) in a bit more than two column volumes of re-equilibration (so-called “repeatable equilibrium”) [20]. Given a stable column and a good signal to noise ratio [8] the parameters that control retention time precision are: temperature ( T ), initial ( ϕ 0 ) and final ( ϕ f )mobile phase composition, gradient time ( t G ) and flow rate ( F ).…”

“…In prior work we showed that at flow rates of 1 mL/min or less temperature control of the column was not as tight as at higher flow rates (2-3 mL/min). This was attributed to quite evident changes in the duty cycle of the oven column compartment temperature control system [20]. …”

“…gradual changes in solvent composition with time) [25]. Extremely good isocratic retention reproducibility can be achieved by gravimetric metering of the eluent [20]. When an improperly functioning pump is used disturbances in mobile phase composition or flow rate and changes in gradient time have a direct impact on the “gradient steepness” and will change the retention time [43-45].…”