Confirmation of Separation Mechanism Through Visualization of Microfluidic Behavior of Fluorescent Analytes in Tube Radial Distribution Chromatography

A method of tube radial distribution chromatography (TRDC) based on an annual flow with inner and outer phases created through phase transformation in an open-tubular capillary was developed. The outer phase works as a pseudo-stationary phase under laminar flow conditions in TRDC. Two model fluorescent analytes, hydrophobic perylene and hydrophilic Eosin Y, were separated by TRDC using a water/acetonitrile/ethyl acetate mixed solution (3:8:4 volume ratio, organic solvent-rich or 4:3:1 volume ratio, water-rich) as an eluent solution and a fused-silica capillary tube (75  m inner diameter and 120 cm length/100 cm effective length) as a separation column. We observed for the first time microfluidic behavior of fluorescent analytes that distributed and separated in the capillary tube visually by fluorescence microscope-charge coupled device camera. The separation mechanism in the TRDC was confirmed with visual fluorescence data and the obtained chromatographic data with UV detection. Furthermore, the elution times of perylene and Eosin Y were calculated based on their retention factors for the two phases and laminar flow conditions. The data and results obtained support the proposed separation mechanism in