Close Elizabeth D, Nwokeoji Alison O, Milton Dafydd, Cook Ken, Hindocha Darsha M, Hook Elliot C, Wood Helen, Dickman Mark J
Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK.
Thermo Fisher Scientific, Stafford House, Boundary Way, Hemel Hempstead HP2 7GE, UK.
J Chromatogr A. 2016 Apr 1;1440:135-144. doi: 10.1016/j.chroma.2016.02.057. Epub 2016 Feb 23.
Ion pair reverse-phase liquid chromatography has been widely employed for nucleic acid separations. A wide range of alternative stationary phases have been utilised in conjunction with ion pair reverse-phase chromatography, including totally porous particles, non-porous particles, macroporous particles and monolithic stationary phases. In this study we have utilised superficially porous silica particles in conjunction with ion pair reverse-phase liquid chromatography for the analysis of nucleic acids. We have investigated a range of different pore-sizes and phases for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and RNA. The pore size of the superficially porous silica particles was shown to significantly affect the resolution of the nucleic acids. Optimum separations of small oligonucleotides such as those generated in RNase mapping experiments were obtained with 80Å pore sizes and can readily be interfaced with mass spectrometry analysis. Improved resolution of larger oligonucleotides (>19mers) was observed with pore sizes of 150Å. The optimum resolution for larger dsDNA/RNA molecules was achieved using superficially porous silica particles with pore sizes of 400Å. Furthermore, we have utilised 150Å pore size solid-core particles to separate typical impurities of a fully phosphorothioated oligonucleotide, which are often generated in the synthesis of this important class of therapeutic oligonucleotide.
离子对反相液相色谱已被广泛用于核酸分离。多种替代固定相已与离子对反相色谱结合使用，包括全多孔颗粒、无孔颗粒、大孔颗粒和整体固定相。在本研究中，我们将表面多孔硅胶颗粒与离子对反相液相色谱结合用于核酸分析。我们研究了一系列不同孔径和固定相，用于分析多种核酸，包括寡核苷酸、寡核糖核苷酸、硫代磷酸寡核苷酸以及高分子量双链DNA和RNA。结果表明，表面多孔硅胶颗粒的孔径对核酸的分离度有显著影响。对于小的寡核苷酸，如核糖核酸酶图谱实验中产生的那些，使用80Å孔径可获得最佳分离效果，并且可以很容易地与质谱分析联用。对于较大的寡核苷酸（>19聚体），观察到使用150Å孔径时分离度有所提高。对于较大的双链DNA/RNA分子，使用400Å孔径的表面多孔硅胶颗粒可实现最佳分离度。此外，我们还使用150Å孔径的实心核颗粒分离了完全硫代磷酸化寡核苷酸的典型杂质，这些杂质在这类重要的治疗性寡核苷酸合成过程中经常产生。
