Separation Behavior of Short Oligonucleotides by Ion-Pair Reversed-Phase Capillary Liquid Chromatography Using a Silica-Based Monolithic Column Applied to Simple Detection of SNPs

Abstract

The separation behaviors of short oligonucleotides using capillary liquid chromatography have been investigated comparatively on an octadecylsilane (ODS) packed column (0.3 mm inner diameter, particle size 3 μm) and a monolithic ODS column (0.5 mm inner diameter, through pore size 2 μm). The present paper clearly demonstrates that the resolution obtained using the monolithic ODS packed column is much higher than that using a semi-μ-high-performance liquid chromatography (HPLC) column on a semi-μ-HPLC system. Furthermore, it was surprising that complete baseline resolution of oligonucleotides, typically shorter than 30-mer, with single nucleotide units was achieved using the monolithic column, although this was not possible using the ODS packed column. Good separation of short oligonucleotides was achieved at 40–60 °C with a linear 40–60 % gradient of methanol content. Separation conditions on the monolithic column were optimized regarding the type of counterion, methanol content, temperature, and flow rate. Using the present method, detection of single-nucleotide polymorphisms was achieved without using a mass-spectrometry (MS) detector.