The continuous development of new chemicals enhances the complexity of environmental analysis and poses a risk to environmental and human health. Awareness is increasing that together with the chemical products on the market, the enormous number of transformation and by-products may contribute to this risk. Effect-directed analysis (EDA) has been developed to identify major toxicants in such complex mixtures. Separation techniques in EDA are applied to reduce the complexity of environmental mixtures and provide valuable information on physicochemical and thus structural properties of candidate toxicants. Within the last decades, separation science provided an extensive understanding of processes and mechanisms in chromatography, developed novel stationary phases with specific separation properties, and introduced modeling tools such as linear solvation energy relationships (LSER) to predict retention. A selection of these tools is compiled in the present paper to support the exploitation of present knowledge on chromatography to enhance the ability to identify so far unknown toxicants in complex mixtures.