ArticleAnalytical and bioanalytical chemistry2026
Screening and optimization of online comprehensive two-dimensional liquid chromatography conditions for the analysis of hospital wastewater.
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Abstract
Pharmaceutical residues in freshwater systems constitute a growing environmental problem. An important point source of these pharmaceuticals is hospital wastewater. The characterization of hospital wastewater is challenging because of its complex nature. Pharmaceuticals and their metabolites display a large variety of physicochemical properties, while matrix compounds create additional complexity. Innovative analytical approaches are hence needed to characterize these challenging samples. A promising technique is online comprehensive two-dimensional liquid chromatography (LC × LC), combining two orthogonal separation modes to increase the separation power significantly. Because of the many optimization parameters involved, method development in online LC × LC is complicated. It is difficult to predict which combinations will result in the highest peak capacity for a specific sample. In this work, different separation systems are evaluated for the online LC × LC analysis of pharmaceuticals, using an in-house developed Python-based 2D combination selector (PCS) tool. Practical peak capacities of different combinations, determined using an orthogonality score based on 12 different orthogonality metrics and predicted peak capacities, are used to select promising LC × LC conditions, including reversed-phase (RPLC) and hydrophilic interaction (HILIC) LC. Three promising combinations are further optimized, with special focus on their mobile phase incompatibility. To deal with these incompatibility issues, both active solvent modulation and flow splitting are investigated. After optimization, the RPLC × RPLC method displays the best
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