Design of Experiments-Based Development of UHPLC Methods for Targeted Analysis of Phytoplankton Pigments and Phycocyanin in Aquatic Samples: Characterization for Chemotaxonomic and Remote Sensing Applications

The accurate quantification of phytoplankton pigments is critical for understanding aquatic ecosystem dynamics, supporting satellite algorithm validation, and advancing chemotaxonomic and biodiversity studies. This doctoral thesis shows the development, optimization, and validation of two independent Ultra-High Performance Liquid Chromatography (UHPLC) methods for pigment analysis in marine and inland water samples, both guided by a Design of Experiments (DoE) strategy. The first method addresses the need for a more efficient and environmentally sustainable analysis of phytoplankton pigment diversity. A novel ethanol-based UHPLC method using a core-shell C8 chromatographic column was developed to resolve 33 ecologically relevant pigments, including challenging pairs such as divinyl/monovinyl chlorophylls and xanthophyll isomers. The optimization, based on a central composite DoE design, enabled full resolution in under 26 minutes, reducing both toxic solvent use and analysis time. The method was validated on standards, algal cultures, and natural samples from the northwestern Adriatic Sea. The second UHPLC method targets the quantification of phycocyanin (C-PC), a key pigment in cyanobacteria, extracted from concentrated filter-based samples—commonly collected in satellite validation campaigns. A fullfactorial DoE was applied to compare extraction conditions, optimize chromatographic separation on a reversed-phase C5 chromatographic column using an acetonitrile gradient, and validate the method on natural samples and algal cultures for sensitivity, accuracy, and reproducibility. The result is a fast and robust UHPLC protocol suitable for routine monitoring of cyanobacterial blooms and satellite product validation. Together, these analytical advancements contribute to the development of more reliable tools for aquatic pigment quantification, offering critical support for satellite remote sensing calibration, phytoplankton functional type discrimination, and environmental monitoring programs.