Abstract
This work describes the application of a pressure-enhanced liquid chromatography (PE-LC) setup to tune the separation of various large molecules comprised of nucleic acids (oligonucleotides, messenger ribonucleic acid [mRNA], and deoxyribonucleic acid [DNA]). When adding pressure as a method development parameter, it indeed becomes possible to modify retention, selectivity, and peak width. As an example, the separation of oligonucleotides having sizes comprised between 40 and 100-mer in ion-pairing reversed-phase liquid chromatography (IP-RPLC) was drastically improved by using a stepwise pressure gradient to selectively shift the retention of a peak or group of peaks. Resolution was increased from 1.5 to 11.8 when setting a rapid high pressure step (twofold increased pressure) during the run. On the other hand, it was also possible to improve the separation of erythropoietin (EPO) mRNA and related impurities under ion-exchange chromatography (IEX). However, with this biomolecule, the best separation was achieved by reducing the pressure in the system. Finally, for another sample (DNA ladder) under IEX conditions, the pressure was found to have a limited impact on the overall selectivity. As highlighted in this work, pressure is an additional parameter that can be successfully used to develop LC methods of large biomolecules.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
