Counter-Current Chromatographic Separation of Nucleic AcidConstituents with a Polar Volatile Organic-Aqueous Two-Phase SolventSystems with ELSD Detection

摘要

Highly hydrophilic volatile organic/aqueous two-phase solvent systems containing an organic salt such as,acetonitrile /800 mM and 1200 mM ammonium acetate (1 : 1, v/v) were efficiently utilized for high-speed counter-currentchromatography (HSCCC) to separate hydrophilic compounds. The retention of the upper and the lower stationary phasesin the column of the cross-axis coil planet centrifuge (CCC instrument) was studied by changing the flow-rate of themobile phase (1.0–3.0 ml/min). Using the acetonitrile/800 mM ammonium acetate two-phase solvent system, thestationary phase was retained at 46.3% relative to the total column capacity of 65 ml by the reversed-phase elution modeat a flow-rate of 1.0 ml/min. The best retention of the stationary upper phase of 51.5% was obtained by the solvent systemof the acetonitrile/1200 mM ammonium acetate at the above flow-rate. With the acetonitrile/800 mM ammonium acetatesystem the base line separation of adenine and adenosine monophosphate (AMP) detected by evaporative light scatteringdetector (ELSD) and UV was achieved with lower phase mobile at a flow-rate of 1.0 ml/min within 70 min.