Homogeneous liquid–liquid extraction as an alternative sample preparation technique for biomedical analysis

Abstract Liquid‐liquid extraction is a widely used technique of sample preparation in biomedical analysis. In spite of the high pre‐concentration capacities of liquid–liquid extraction, it suffers from a number of limitations including time and effort consumption, large organic solvent utilization, and poor performance in highly polar analytes. Homogeneous liquid–liquid extraction is an alternative sample preparation technique that overcomes some drawbacks of conventional liquid–liquid extraction, and allows employing greener organic solvents in sample treatment. In homogeneous liquid–liquid extraction, a homogeneous phase is formed between the aqueous sample and the water‐miscible extractant, followed by chemically or physically induced phase separation. To form the homogeneous phase, aqueous samples are mixed with water‐miscible organic solvents, water‐immiscible solvents/cosolvents, surfactants, or smart polymers. Then, phase separation is induced chemically (adding salt, sugar, or buffer) or physically (changing temperature or pH). This mode is rapid, sustainable, and cost‐effective in comparison with other sample preparation techniques. Moreover, homogeneous liquid–liquid extraction is more suitable for the extraction of delicate macromolecules such as enzymes, hormones, and proteins and it is more compatible with liquid chromatography with tandem mass spectrometry, which is a vital technique in metabolomics and proteomics. In this review, the principle, types, applications, automation, and technical aspects of homogeneous liquid–liquid extraction are discussed.