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J Chromatogr A. 2016 Apr 1;1440:135-144. doi: 10.1016/j.chroma.2016.02.057. Epub 2016 Feb 23.

Nucleic acid separations using superficially porous silica particles.

Author information

1
Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK.
2
Thermo Fisher Scientific, Stafford House, Boundary Way, Hemel Hempstead HP2 7GE, UK.
3
GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.
4
Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK. Electronic address: m.dickman@sheffield.ac.uk.

Abstract

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.

KEYWORDS:

Ion pair reverse-phase chromatography; Nucleic acids; Oligonucleotides; RNA

PMID:
26948761
PMCID:
PMC4801196
DOI:
10.1016/j.chroma.2016.02.057
[Indexed for MEDLINE]
Free PMC Article

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