BackgroundIn the past three decades, liquid chromatography (LC) has been recognized as a significant environmental, health, and safety burden due to its heavy reliance on toxic organic solvents. Various chromatographic modes are in vogue today for complex analyses, such as sub/supercritical fluid chromatography (SFC) and enhanced fluidity liquid chromatography (EFLC). These modes are often advertised as universally green compared to the traditional all-liquid reversed (RPLC) and normal phases (NPLC). Quantitative greenness evaluations must be done to validate or invalidate this assumption and allow separation scientists to make educated choices when deciding on what mode to use. ResultsIn this work, we modify the Analytical Method Greenness Score () to include the cycle time of the instrument, and with the help of the first-order mathematical optimality condition ( gradient = 0), we show that SFC and EFLC are not always the greenest option as they are often thought to be. Most of the greenness metrics have ignored the cycle time of instruments, yet this key component changes the entire response to flow rate. The complex case of separating tobacco alkaloid enantiomers (nicotine, nornicotine, anabasine, and anatabine) was selected as an illustrative environment for comparing and contrasting separation modes using the modified greenness metric. These enantiomers have been selected due to their notorious difficulty in separation over the past 30 years. Using this family of molecules, four unique retention patterns were observed covering a wide variety of retention phenomena seen in small molecule enantioseparations. SignificanceThe modified metric will assist practicing analytical chemists in assessing the environmental impact of their separation methods from a single run in a given chromatographic mode. The proposed methodology identifies the minimum score corresponding to the greenest separation to be adapted for routine chemical analysis.