A comprehensive study on the phenomenon of total breakthrough in liquid chromatography.

Differences in elution strength between the sample solvent and the mobile phase usually give rise to undesirable effects on the chromatographic separation, which may range from slight broadening to severe peak deformation or even splitting. In the most extreme case, the retention factor of the analyte at the head of the column is so small at the time of injection that part of the analyte goes through the column with very little interaction with the stationary phase and hence elutes very close to the column dead time. This phenomenon is known as breakthrough. Usually, during breakthrough, the retained peak displays a wide array of deformations and it is not rare that multiple retained peaks appear for a given injected analyte. However, under certain conditions, it has been demonstrated that these deleterious effects could fully disappear, leaving only one breakthrough peak and one symmetrical peak on the chromatogram. This so-called "total breakthrough" phenomenon was recently highlighted in the specific context of the 2D-LC separation of peptides but has yet to be explained. In the present paper, we describe the results of a comprehensive study aiming to better understand and define the conditions of emergence of both breakthrough and total breakthrough phenomena in liquid chromatography. The effects of a broad range of parameters, including the nature of the solute, the retention mechanism, the injection and elution conditions, the column temperature, and the injected sample concentration on the occurrence of both phenomena were investigated. While breakthrough was found to occur for all studied compounds, it appears that the presence of positive charges on the molecule is a prerequisite for observing a total breakthrough phenomenon. Among all the parameters investigated in this work, only the injection conditions and the analyte retention were found to be impactful on the onset of both phenomena. This finding allowed us to suggest one necessary and sufficient condition, relying on the injection of critical volumes to observe each respective phenomenon. These critical volumes only depend on the column dead volume and the retention factor of the analyte in the injection solvent.