Separation of nucleobases and their derivatives with organic-high ionic strength aqueous phase systems by spiral high-speed counter-current chromatography

A set of nucleic acid constituents were separated with ultra polar two-phase solvent systems by a spiral multilayer coil mounted on the rotary frame of a type-J coil planet centrifuge. These two-phase systems were composed of 1-butanol/ethanol/50% saturated aqueous ammonium sulfate at various volume ratios. Nucleobases including adenine, cytosine, uracil, and thymine; nucleosides including adenosine, guanosine, cytidine, and uridine; and nucleotides including, AMP, GMP, CMP, UMP, and TMP are partitioned in each group with suitable solvent ratios. Adenine derivatives such as adenosine, AMP, ADP, and ATP were well resolved in the most polar solvent system composed of ethanol/50% saturated aqueous ammonium sulfate at a volume ratio of 1:2. It was found that cytosine and cytidine peaks showed some irregular two peaks probably due to their keto and enol isomers, while the separation of AMP forms two peaks especially when TMP was added in the sample solution, the mechanism of which is now under investigation in our laboratory.