Slow equilibration of reversed-phase columns for the separation of ionized solutes

2008

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.

Section: Introductionmentioning

confidence: 99%

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

Schellinger

Stoll

2008

“…Also, the study by Marchand et. al has shown that the Zorbax SB-C 18 column showed minimal retention drift under isocratic conditions [8]. However, we believe that the equilibration processes under isocratic and gradient conditions are fundamentally different.…”

Section: Introductionmentioning

confidence: 66%

“…eluent pH, degree of solute ionization and stationary phase type) [8]. Clearly, if such slow column reequilibration were observed under gradient conditions it would drastically increase the cycle time.…”

Section: Introductionmentioning

confidence: 99%

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

Schellinger

Stoll

2008

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.

“…We next measured the actual fluctuations in a specific instrument and computed the contribution to the retention times for three distinct sets of solutes. Other instrumental factors impacting retention precision such as electronic noise in the peak signal [8-11], and long term drift [12] have been studied elsewhere.…”

Section: Introductionmentioning

confidence: 99%

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

Beyaz

Fan

et al. 2014

The precision of retention time in RPLC is important for compound identification, for setting peak integration time windows and in fundamental studies of retention. In this work, we studied the effect of temperature (T), initial (ϕ0) and final mobile phase (ϕf)composition, gradient time (tG), and flow rate (F) on the retention time precision under gradient elution conditions for various types of low MW solutes. We determined the retention factor in pure water (k′w) and the solute-dependent solvent strength (S) parameters of Snyder's linear solvent strength theory (LSST) as a function of temperature for three different groups of solutes. The effect of small changes in the chromatographic variables (T, ϕ0, ϕf, tG and F) by use of the LSST gradient retention equation were estimated. Peaks at different positions in the chromatogram have different sensitivities to changes in these instrument parameters. In general, absolute fluctuations in retention time are larger at longer gradient times. Drugs showed less sensitivity to changes in temperature compared to relatively less polar solutes, non-ionogenic solutes. Surprisingly we observed that fluctuations in temperature, mobile phase composition and flow rate had less effect on retention time under gradient conditions as compared to isocratic conditions. Overall temperature and the initial mobile phase composition are the more important variables affecting retention reproducibility in gradient elution chromatography.