Benchmarking green analytical methods for phytoplankton pigments extraction and quantification

Phytoplankton pigments, including chlorophylls, carotenoids, and xanthophylls, are key biomarkers for assessing aquatic ecosystem productivity, biodiversity, and for validating satellite-based ocean color products. High-performance liquid chromatography (HPLC) remains the reference method for pigment analysis due to its sensitivity and resolution, but conventional workflows rely on organic solvents and energy-intensive procedures that conflict with sustainability objectives. This study systematically evaluates the environmental performance of twelve representative chromatography methods for phytoplankton pigments analysis using a multi-metric assessment framework. Greenness was quantified with complementary tools, including the Analytical Eco-Scale (ESA), ChlorTox, AGREE/AGREEprep, AMGS, ComplexMoGAPI, BAGI, and RGB 12, capturing solvent toxicity, energy consumption, waste generation, and practical applicability. Results indicate a progressive shift toward greener methodologies, with UPLC methods achieving the highest scores across multiple metrics and reduced chemical hazard, closely followed by supercritical fluid chromatography (SFC). SFC was scarcely implemented and limited by the quantification of fewer compounds, looks promising. Despite improvements, methodological constraints—such as sample preservation, batch processing for quality assurance, and filtration-based extraction—limit full compliance with Green Analytical Chemistry (GAC) principles. Trade-offs among evaluation frameworks highlight the importance of multi-metric assessment, with AGREEprep providing detailed insights into sample preparation energy and waste, while ChlorTox, BAGI, and RGB 12 offer complementary perspectives. These findings demonstrate the potential for integrating greener solvents, UPLC, and Design of Experiments (DoE) approaches to optimize both environmental sustainability and analytical performance. Adoption of such strategies in future method development and inter-calibration exercises will support reliable pigment quantification while aligning analytical workflows with sustainability targets, including United Nations Sustainable Development Goals and environmental monitoring directives.