Tube radial distribution chromatography system developed by combining commercially available HPLC system and open-tubular capillary tube as separation column.

Tube radial distribution chromatography based on tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported, where the annular flow is created through phase separation multiphase flow. The chromatographic system requires some specific instruments and treatments for microfluidic flow in the capillary tube. In this study, we developed a new set-up for tube radial distribution chromatography by combining a commercially available HPLC system with an open-tubular capillary tube (with an inner diameter of 100 µm) as a separation column instead of a conventional packed column. The analyte solution was injected with an injection valve (2 µL volume) and a ternary solution of water/acetonitrile/ethyl acetate (3:8:2 vol ratio) was delivered as the eluent to the capillary tube at a flow rate of 8.6 µL min-1. The chromatographic system, that is, the HPLC system equipped with the open-tubular capillary tube, could successfully separate the model analytes, 1-naphthol, 1-naphthalenesulfonic acid, and 2,6-naphthalenedisulfonic acid, with base-line separation. The inner and outer phases in the annular flow worked as the mobile and pseudo-stationary phases, respectively, in the tube radial distribution chromatography system. The experimentally obtained elution times of the analytes were compared with their corresponding theoretical values calculated using their capacity factors for the inner and outer phases and the linear flow velocities of the respective phases.

Implementation of Tube Radial Distribution Chromatography by Using a Commercially Available HPLC System.

Tube radial distribution chromatography based on tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported. The chromatographic system requires specific instruments and treatments for microfluidic flow in the capillary tube. In this study, we have developed a new model of tube radial distribution chromatography, which is comprised of a commercially available HPLC system without any packed separation columns. Separation is performed in an open-tubular pipe (100-µm inner diameter and 350-cm length; temperature, 5°C) connected between the pump and the detector in the HPLC system. An analyte solution is introduced with a sample injector (2-µL volume) and a ternary water/acetonitrile/ethyl acetate mixed solution (volume ratio of 3:8:2) is delivered as an eluent solution into the pipe at a flow rate of 10-µL min-1. Fused silica and stainless pipes can separate 1-naphthol and 2,6-naphthalenedisulfonic acid, but a polyetheretherketone pipe cannot. The obtained data provides an important clue to practical developments in separation science.

Protein separation through preliminary experiments concerning pH and salt concentration by tube radial distribution chromatography based on phase separation multiphase flow using a polytetrafluoroethylene capillary tube.

Protein mixtures were separated using tube radial distribution chromatography (TRDC) in a polytetrafluoroethylene (PTFE) capillary (internal diameter=100µm) separation tube. Separation by TRDC is based on the annular flow in phase separation multiphase flow and features an open-tube capillary without the use of specific packing agents or application of high voltages. Preliminary experiments were conducted to examine the effects of pH and salt concentration on the phase diagram of the ternary mixed solvent solution of water-acetonitrile-ethyl acetate (8:2:1 volume ratio) and on the TRDC system using the ternary mixed solvent solution. A model protein mixture containing peroxidase, lysozyme, and bovine serum albumin was analyzed via TRDC with the ternary mixed solvent solution at various pH values, i.e., buffer-acetonitrile-ethyl acetate (8:2:1 volume ratio). Protein was separated on the chromatograms by the TRDC system, where the elution order was determined by the relation between the isoelectric points of protein and the pH values of the solvent solution.