Multicomponent (n >or= 3) sorption isotherms in reversed-phase liquid chromatography: the effect of immobilized eluent on the retention of analytes.

The experimental technique of tracer pulse chromatography was used to simultaneously measure the uptake of eluent components by a C(18)-bonded reversed-phase liquid chromatography (RPLC) packing and the retention factors for a series of test analytes over the full range of eluent composition for methanol and acetonitrile with water. The primary objective of the research was to determine whether or not the uptake of eluent components immobilized as part of the stationary phase would influence the retention of analyte standards. Both acetonitrile and methanol were absorbed in or adsorbed on the C(18)-bonded phase with the maximum amount of acetonitrile sorbed being about four times that of methanol. The thermodynamic void volume of the column and the excess sorption isotherms of acetonitrile, methanol, and water in binary aqueous/organic mixtures were determined directly from the tracer pulse experiments. The absolute sorption isotherms of the eluent components were indirectly estimated by a combination of techniques. Regression analysis of the nonstationary inflection point of the excess isotherms provided an estimate of the volume of eluent sorbed by the stationary phase but only over a limited eluent composition range. In order to expand the applicable composition range, several commonly used "unretained" probe solutes were tested to determine the accuracy of the assumption that the retention volumes of these solutes provided a viable measure of the kinetic void volume (mobile-phase volume) of the column. The difference between the thermodynamic and kinetic void volumes provided an estimate of the absolute volume of eluent present in the stationary phase. The experimental results showed that some solutes, viz., water and thiourea, did provide an accurate measure of the mobile-phase volume but only over a limited range of eluent composition. Using deuterated water as the unretained dead time marker for water-rich eluents combined with the regression results from excess isotherm data, the absolute volume of eluent sorbed by the stationary phase could be estimated over the full range of eluent composition. The effect of this uptake of eluent on the retention of the test solutes appeared to be minimal for this particular set of test analytes.