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 (ϕo) 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, ϕo, 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 most important variables affecting retention reproducibility in gradient elution chromatography.
Bibliographical noteFunding Information:
This work was funded by the National Institutes of Health Grant GM 054585 and the National Institute of Justice , through the Midwest Forensic Research Center at Ames Laboratory. We also acknowledge Agilent Technologies for the donation of the columns. Abbott Laboratories (now AbbVie) are thanked for partial support of the work.
- Gradient elution parameters
- Gradient elution vs isocratic elution
- HPLC instrument parameters
- Linear solvent strength theory
- Pharmaceutical compounds
- Retention time precision