[en] The growing demand for efficient and selective separation of complex mixtures in both industrial and
academic contexts underscores the need for a better understanding of compound class behavior in
liquid chromatography. Generally, the optimal choice of stationary phase for separating specific classes
of compounds is unclear, particularly when dealing with chemically diverse analytes. To address this
challenge, this study evaluates and compares the elution and separation performance of a broad range
of analytes using four distinct UHPLC stationary phases: Allure™ Silica (bare silica), Ultra® Amino
(amino-bonded silica), Nucleosil® Chiral-2 (donor-acceptor complex chromatography, DACC), and
Inertsil® Diol (diol-bonded silica). The Megamix standard mixture, consisting of 76 compounds from
diverse aliphatic, aromatic, and heteroatomic classes, was used for the analysis. These compounds
exhibit a wide range of polarities, aromatic characteristics, and functional groups. Elution profiles on
the different columns were assessed to identify trends in retention behavior, selectivity, and class
based resolution. The Allure™ Silica column demonstrated strong selectivity for polar compounds,
effectively clustering them by chemical composition, but showed prolonged retention of highly polar
analytes, leading to longer run times. The Nucleosil® Chiral-2 column offered excellent separation of
aromatic compounds according to their number of aromatic rings, largely due to strong electron donor
acceptor (EDA) interactions linked to HOMO energy. However, this column showed reduced
performance for non-aromatic compounds and partial overlap between classes, influenced by
alkylation-related retention. The Inertsil® Diol column exhibited moderate polarity and retained
oxygenated and chloro-oxygenated compounds effectively. It enabled fine intra-class separation among
polar compounds and displayed the highest repeatability of all tested columns. In contrast, the Ultra®
Amino column, with higher polarity than Diol and improved chemical stability over bare silica, provided
good separation of oxygenated compounds from others but lacked selectivity for less polar compounds
and had lower repeatability than Diol.
