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.
Highlights
We have investigated a range of different pore-sizes and phases (C18, C4) for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and ribonucleic acids (RNA)
Typical peak widths of 0.12 min were obtained on the 80 Å compared to 0.07 min on the 150 Å pore size solid-core particles, demonstrating that increased resolution of the oligonucleotides were obtained on the 150 Å pore size solid-core particles at a flow rate of 0.4 ml/min
The results of the oligonucleotide separations using the 150 Å pore size solid-core particles demonstrate that rapid, high resolution separation of oligonucleotides can be achieved in a similar way to those obtained using non-porous particles and UPLC applications
Summary
Ion pair reverse-phase chromatography (IP RP HPLC) has been widely employed for nucleic acid separations [1,2,3]. A wide range of reverse-phase stationary phases have been utilised for IP RP HPLC including traditional totally porous C18 particles (silica and polymeric stationary phases) [1,2,3,4,5]. The column media are robust, being resistant to a broad range of temperatures and pH This has led to a wide variety of developments in the analysis of nucleic acids using IP RP HPLC, including the sequence-independent sizing of duplex DNA [9], the analysis of oligonucleotides and the separation and purification of singlestranded (ss) DNA [10,12]. The advantages of core–shell particle columns for rapid HPLC analysis of biomolecules including proteins and nucleic acids has been demonstrated, attributing this improved performance to superior mass transfer kinetics [28,29]. We have investigated a range of different pore-sizes and phases (C18, C4) for the analysis of a diverse range of nucleic acids including oligonucleotides, oligoribonucleotides, phosphorothioate oligonucleotides and high molecular weight dsDNA and RNA
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