Pressure–Enhanced Liquid Chromatography as a Suitable Approach to Improve Selectivity for Large Molecule Separations

This work describes the application of a pressure-enhanced liquid chromatography (PE-LC) setup to tune separation various large molecules comprised nucleic acids (oligonucleotides, messenger ribonucleic acid [mRNA], and deoxyribonucleic [DNA]). When adding pressure as method development parameter, it indeed becomes possible modify retention, selectivity, peak width. As an example, oligonucleotides having sizes between 40 100-mer in ion-pairing reversed-phase (IP-RPLC) was drastically improved by using stepwise gradient selectively shift retention or group peaks. Resolution increased from 1.5 11.8 when setting rapid high step (twofold pressure) during run. On other hand, also improve erythropoietin (EPO) mRNA related impurities under ion-exchange (IEX). However, with this biomolecule, best achieved reducing system. Finally, for another sample (DNA ladder) IEX conditions, found have limited impact on overall selectivity. highlighted work, is additional parameter that can be successfully used develop LC methods biomolecules.