Temperature has been considered as an important variable in Liquid Chromatography (LC).Operating at higher temperatures has led to "High Temperature Liquid Chromatography" which has attracted major attention during the last years since an increased chromatographic performance and a reduced back pressure can be achieved [1].In addition, higher temperatures also cause reduced retention properties in reversed-phase mode.This allows a reduction of the organic phase content of the mobile phase resulting in a "greener" and more cost effective approach.However, the amount of reports where the effect of decreased temperatures (sub-zero) and possible accompanying applications are investigated have been very limited.