Novel aqueous two-phase systems based on deep eutectic solvent and ionic liquid for green extraction of chlorophyll from mulberry leaves: A comparative study on phase behavior and extraction efficiency

Chlorophyll is a valuable natural pigment with wide applications in the food, pharmaceutical, and cosmetic industries, creating a growing demand for efficient and environmentally conscious extraction methods. In this study, chlorophyll extraction from mulberry leaves using conventional solid-liquid extraction with ethanol was compared with a series of aqueous biphasic systems (ATPSs) based on polypropylene glycol 400 (PPG400). The investigated ATPSs were prepared by combining PPG400 with either three choline chloride-based deep eutectic solvents (DES) containing glycerol, lactic acid, or acetic acid as hydrogen bond donors, or a cholinium acetate ionic liquid (IL). The phase behavior and extraction performance of these systems were systematically evaluated under identical extraction conditions (25 °C for 1 h), and chlorophyll content was determined by UV–Vis absorption spectrophotometry. Conventional ethanol extraction yielded an average chlorophyll content of 1.28 mg g −1 dry biomass, corresponding to a relative extraction efficiency of 49.60%. Among all investigated systems, the ATPS composed of 20 wt% cholinium acetate and 40 wt% PPG400 showed the highest extraction performance, yielding 2.58 mg g −1 dry biomass at a solid-liquid ratio of 1:100. This system was used as the reference for calculating the relative extraction efficiency; therefore, its extraction efficiency was defined as 100% under the investigated experimental conditions. Among the DES-based ATPSs, the system containing 22 wt% choline chloride/glycerol and 43 wt% PPG400 provided the best performance, with a chlorophyll yield of 2.05 mg g −1 dry biomass and a relative extraction efficiency of 79.80%. Although the choline chloride/lactic acid-based system exhibited the strongest phase separation, its extraction performance was comparatively lower (approximately 1.50 mg g −1 dry biomass and 58–60% relative extraction efficiency), indicating that phase separation alone is not sufficient to predict extraction performance. Instead, the observed extraction behavior is likely governed by the combined effects of phase equilibrium, solvent properties, and specific solvent–solute interactions. To the best of our knowledge, this is the first comparative study evaluating DES and IL based aqueous biphasic systems combined with PPG400 for chlorophyll extraction from mulberry leaves under identical experimental conditions, providing a systematic comparison of their phase behavior and extraction performance. Overall, the cholinium acetate/PPG400 ATPS demonstrated superior chlorophyll extraction under mild operating conditions and represents a promising aqueous biphasic platform for chlorophyll recovery from plant biomass. Although the developed system exhibits several characteristics associated with greener extraction processes, further studies on solvent recyclability, quantitative green metrics, and life-cycle assessment are needed to comprehensively evaluate its environmental sustainability.