Column “dead volume” in liquid chromatography

Abstract

The physical and chromatographic volumes associated with a liquid chromatography column have been considered theoretically and their inter-relationship is discussed. The two chromatographically important dead volumes, the kinetic dead volume and the thermodynamic dead volume, have been identified and it is shown that, whereas the kinetic dead volume (the volume of the moving phase) was common to all solutes, the thermodynamic dead volume was unique to each solute. The solute dependence of the thermodynamic dead volume resulted from any exclusion characteristics that the stationary phase possessed. Stationary phases based on silica exhibited strong exclusion properties and hence the thermodynamic dead volume for silica-based phases was strongly dependent on solute size. It followed that the pertinent thermodynamic dead volume had to be determined strictly for each solute. It also followed that correlation of retention with solvent composition could not be carried out employing k′(solute capacity factor) values, as these were dependent on accurate dead volume measurements. It is recommended that for such a correlation, corrected retention volumes are employed and then only where the solute is eluted at sufficiently high k′ values. Under such circumstances any errors that would result from incorrect dead volume measurements are minimised. Experimental methods are given for the evaluation of all chromatographically pertinent volumes and, as an example, the procedures were applied to a Zorbax C₈ reversed-phase column. The data obtained are discussed in the light of the theory presented.