Behaviour of Macromolecular RNA in Reversed-Phase HPLC

Abstract

Large RNA molecules are retained on reversed-phase columns and can be eluted by increasing the concentration of acetonitrile. The retention behaviour depends on the base composition but not on the length of the chain, thereby providing a means for the separation of RNA molecules with different base composition. However, isocratic elution is not feasible due to the sudden changes in retention characteristics which are observed with slight variations in acetonitrile concentration. In order to interpret these results, a model has been developed called ‘multiple-point interaction theory’, based on the assumption that macromolecules are flexible and very large as compared to the hydrophobic phase. This model fits the experimental data and could be applied to all types of flexible macromolecules, especially proteins and nucleic acids, when they are chromatographed on reversed-phase columns. In the case of RNA, it has been observed that the retention time depends on ionic strength, pH, and temperature as expected for a true partition between two phases. Furthermore, an artifact called the ‘polypeak phenomenon’ appears at high flow rates which decreases the resolution of RNA. This polypeak phenomenon is controlled by the slope of the gradient dφ/dv.