Reproducibility of low and high concentration data in reversed-phase liquid chromatography. II. Overloaded band profiles on Chromolith-C18.

Single-component adsorption isotherm data were acquired by frontal analysis (FA) for six low molecular weight compounds (phenol, aniline, caffeine, o-toluidine, p-toluidine and propylbenzoate) on one Chromolith-C18 column (#30, Merck, Darmstadt, Germany), using different methanol:water solutions (composition between 60/40 and 15/85 v/v, depending on the solute) as the mobile phase. These data were modeled for best agreement between the experimental data points and the adsorption isotherm model. The adsorption-energy distributions were also derived and used for the selection of the best isotherm model. Widely different models were obtained for these six compounds, four being convex upward (i.e., Langmuirian) and two having at least one inflection point. Overloaded band profiles corresponding to two different sample sizes (a low and a high loading factor) were recorded on six monolithic columns (#30-35) belonging to the same manufactured lot. These experimental band profiles were compared to the profiles calculated from the isotherm measured by FA on the first column, using the equilibrium-dispersive (ED) model of chromatography. For four of the six columns (#30, #32, #33, and #35), the reproducibility was better than 5 and 2.5% for the low and the high concentration profiles, respectively. On the other two columns (#31 and #34), the bands showed significant and systematic retention time shifts for all six compounds (with nearly identical band shapes), the relative adsorption being between 6 and 15% stronger on column #31 or between 2 and 7% lower on column #34. These differences seem to be correlated with the differences in the total porosities of these columns, which differ by 3% from columns #31 to #34, the higher porosity column giving the stronger adsorption.