The goal of this work was to understand the variation of apparent efficiency when serially couplingcolumns with identical stationary phase chemistries, but with differences in their kinetic performance.For this purpose, a mathematical treatment was developed both for isocratic and gradient modes to assessthe change in plate numbers and peak widths when coupling arbitrary several columns. To validate thetheory, experiments were also carried out using various mixtures of compounds, on columns packed withdifferent particle sizes, to mimic highly efficient (new, not used) and poorly efficient columns (used onewith many injections). Excellent agreement was found between measured and calculated peak widths.The average error in prediction was about 5% (which may be explained by the additional volume of thecoupling tubes).In isocratic mode, the plate numbers are not additive when the coupled columns possess differentefficiencies, and a limiting plate count value can be calculated depending on the efficiency and length ofthe individual columns. Theoretical efficiency limit can also be determined assuming one column in therow with infinite efficiency.In gradient elution mode, the columns' order has a role (non-symmetrical system). When the lastcolumn has high enough efficiency, the gradient band compression effect may outperform the competingband broadening caused by dispersive and diffusive processes (peak sharpening). Therefore, in gradientmode, the columns should generally be sequentially placed according to their increasing efficiency.