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• A retention increase of macromolecules on IEX column with pressure rise was observed. • Up to 80% increase in retention was measured for isocratic elutions. • Gradient elutions of oligonucleotides, proteins and plasmid DNA showed similar trends. • The observed pressure effect depends on the molecule's size. • The separation resolution changed with different column pressure. Shorter analysis times and greater resolving power are contributing factors for transfer of separation methods from an HPLC to a UHPLC system when performing analysis in biopharmaceutical or clinical research. The effect of pressure on separations in reversed phase chromatography is well described, however such investigations on ion exchange columns were previously not conducted. In this study we describe the effect of pressure on retention properties of proteins, oligonucleotides and plasmid DNA in ion exchange chromatography. Different column inlet pressures were obtained by coupling restriction capillaries with column outlet and performing separations at a constant temperature and mobile phase flow rate. Macromolecules were separated in isocratic mode as well as with various linear gradients of salt concentration at a constant pH value. The measured retention time increase was up to 80% for isocratic and 20% for gradient separations for a 500 bar increase in pressure. The effect of pressure was validated on a separate instrument after few months from initial experiments. The influence of pressure on retention properties seems to be dependent on the size, shape and flexibility of the macromolecule and causes different retention shifts when separating a sample with diverse analytes. Such changes in retention time can sometimes exceed the criteria set by European Pharmacopoeia (Ph. Eur.) for the allowable method adjustment and are thus considered to be a result of a different separation method. Therefore, the pressure effect that follows method transfer from HPLC to UHPLC conditions should not be neglected even for gradient separations in ion exchange chromatography, as the resulting retention change may cause revalidation of the separation method. [ABSTRACT FROM AUTHOR]