Chromatographic analysis and separation of short RNA oligonucleotides with novel liquid chromatography methods

Synthetic oligonucleotides have become increasingly important as part of new developments in the use of antisense and small interfering ribonucleic acid (siRNA) as potential therapies for the treatment of various different diseases. The development of analytical methods for the sensitive and quantitative analysis and separation of oligonucleotides is an essential part for the advancement of this research area. A typical oligonucleotide therapeutic sample is a short RNA-based oligonucleotide with about 21-mer length (~7-8 kDa), and with possible chemical modifications prepared by chemical synthesis using an automated synthesizer. Due to their relatively large sizes compared to typical small-molecule compounds, oligonucleotides can be difficult to analyze with traditional analytical methods. There are many different analytical techniques reported for the analysis and separation of oligonucleotides, including capillary gel electrophoresis (CGE), anion-exchange high performance liquid chromatography (AEX-HPLC), and ion-pair reversed-phase liquid chromatography (IP-RPLC). In these research studies, existing liquid chromatography methods for the separation of oligonucleotides were evaluated for improved separations. New and novel approaches were also explored for more efficient analysis and separation of oligonucleotides.