First proof-of-concept of UC/HILIC for extending the versatility of the current art of supercritical fluid separation

Supercritical Fluid Chromatography (SFC), a high-throughput separation technique, has been widely applied as a promising routine method in pharmaceutical, pesticides, and metabolome analysis in the same way as conventional liquid chromatography and gas chromatography. Unified chromatography (UC), an advanced version of SFC, which applied gradient elution with mobile phase changing continuously from supercritical to subcritical and to liquid states, can further extend the SFC applications. UC mostly applying the popular mobile phase of 95%:5%/Methanol:Water with additives allows to analyze many hydrophilic compounds. However, many of phosphorylated metabolites or multi carboxylic acids show very poor peak shapes or even can't be eluted under UC conditions, thus hampering the UC's metabolome coverage. In this study, we proposed the first proof-of-concept of UC/HILIC, a novel strategy to extend the current UC metabolome coverage by employing an aqueous gradient right after the UC gradient on a single packed column in a single measurement. The proposed method showed significant improvement regarding the chromatographic performance and metabolome coverage, while still maintaining the precision and high throughput in comparison with conventional UC methods.