On-column and gradient focusing-induced high-resolution separation in narrow open tubular liquid chromatography and a simple and economic approach for pico-gradient separation

We have recently obtained extraordinarily high efficiencies and sharp peaks using narrow open tubular (OT) columns for liquid chromatographic separations. On-column focusing is commonly observed in liquid chromatography, but this effect alone could not satisfactorily explain the sharpness of these peaks. In this work we investigated the reasons that could have led to the peak sharpness. We hypothesized initially that analytes confined in a narrow OT column might have significantly reduced analyte diffusivities and the reduced diffusivities consequently resulted in the peak sharpness. This hypothesis was invalidated immediately after we measured the diffusion coefficients and did not notice any noticeable diffusivity increases of the analytes inside such columns. We then designed an experiment and revealed a “re-focusing effect”. Investigation of this re-focusing effect eventually led us to the observation of a gradient focusing caused by the composition difference between the eluent and the sample matrix. It was this gradient focusing that had contributed primarily to the peak sharpness. On the basis of this insightful understanding, we further developed a simple and economic approach to perform pico-gradient narrow open tubular liquid chromatographic separations.