Transfer-volume effects in two-dimensional chromatography: Adsorption-phenomena in second-dimension size-exclusion chromatography

Gradient-elution LC × LC is a valuable technique for the characterization of complex biological samples as well as for synthetic polymers. Breakthrough and viscous fingering may yield misleading information on the sample characteristics or deteriorate separation. In LC × SEC another phenomenon may jeopardize the separation. If the analytes adsorb on the SEC column under the injection-plug conditions, peak splitting may occur. In LC × LC the effluent from the first column is the sample solvent for the analytes injected into the second dimension. If a gradient-elution LC × SEC setup is used (i.e. if reversed-phase gradient-elution LC is coupled to organic SEC and if normal-phase gradient-elution LC is coupled to SEC with a polar solvent), the percentage of weak solvent may be significant, especially at short analysis times. In this case adsorption in the first-dimension-effluent zone on the second-dimension SEC column can become an issue and two peaks – the first eluting in size-exclusion mode and the second undergoing adsorption – can be obtained. The work presented in this paper documents peak splitting in LC × SEC of polymers. The adsorption of the polymer on the size-exclusion column was proven in one-dimensional isocratic runs. The observed effects were modeled and visualized through simulation. Studies on the influence of the transfer volume were carried out. Keeping the transfer volume as small as possible helped to minimize peak splitting due to adsorption.