In this research, promising aqueous two-phase systems (ATPS) containing polyoxyethylene (20) sorbitan monolaurate (Tween 20) as a quite biocompatible nonionic surfactant and different salts (K2HPO4, MgSO4, and Na3Citrate) were prepared and their extractive potential was evaluated. These ATPS possess top and bottom phases well-stocked in surfactant and salt respectively which can offer appealing characteristics such as low interfacial tension, low volatility, and ease of scaling-up. Ternary phase diagrams of ATPS consisting of Tween 20 and three different salts were obtained and the results were successfully fitted to the Merchuk correlation. The salting-out ability of salts were assessed. Also, the tie-line data were correlated with the Othmer-Tobias equation. Afterward, the partitioning behavior of cefazolin as a broad-spectrum antibiotic in the proposed ATPS was studied at three temperatures (298.15, 302.15, and 306.15 K) and the influences of Na3Citrate and MgSO4 on partition coefficients were justified by Hofmeister series. Furthermore, the effect of tie-line length on partitioning of cefazolin in ATPS was investigated. The experimental data of cefazolin partition coefficients indicated the proper potential of the Tween 20–Na3Citrate–water biphasic system for the bioseparation process.

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