Theoretical Aspects and Applications of Aqueous Two‐Phase Systems

Abstract

The aqueous two-phase system (ATPS) is commonly known as a technique that yields high-purity products in a single step. It is particularly advantageous for purifying biomolecules like proteins, nucleic acids, enzymes, viruses etc. Currently, aqueous two-phase extraction (ATPE), i.e., liquid-liquid extraction, involves the transfer of the solute from one aqueous phase to another. In ATPE, for recovery of biomolecules, polymer-polymer and polymersalt type systems are used. The most recent developments with respect to recovery of biomolecules by ATPS are reviewed and discussed, covering the mechanism, which controls the phase formation, the conditions of solute partitioning in ATPS processes, and factors influencing the ATPS including concentration and molecular weight (MW) of polymers, types of salt, pH, and temperature. In addition, also the increasing applications of ATPS for the recovery of high-value bioproducts, the benefits of the ATPS recovery system, and the recent developments of alternative low-cost ATPS are highlighted.

Investigating and Optimizing Insulin Partitioning with Conjugated Au Nanoparticles in Aqueous Two-Phase Systems Using Response Surface Methodology

“…To comprehend the correlation between the partition coefficient of a biomolecule and the pH of the system, it is necessary to consider the nature of the charged amino acid residues on the surface of the biomolecule and electrostatic interactions between the biomolecule and polymers. , The partitioning results (shown in Table ) indicate that at pH 5, insulin partitioned into the dextran-rich phase, whereas AuNPs/insulin partitioned into the PEG-rich phase. However, at a pH above 7, the partitioning of insulin and AuNPs/insulin occurred in the PEG-rich phase.…”

“…Generally, negatively charged biomolecules prefer the upper phase (PEG-rich phase), while positively charged biomolecules partition to the lower phase (dextran-rich phase). When the pH of a biomolecule rises above its isoelectric point (pI), it becomes more negative, resulting in an increase in the partition coefficient. , Changes in pH causes a shift in the phase preference due to variations in insulin surface charge (pI = 5.4), and the magnitude of partitioning depends on surface charges. The decrease in the partitioning coefficient between pH 8 and 9 may be attributed to changes in the insulin molecular structure and its interaction with the aqueous environment, e.g., hydrophobic interactions. , The conjugation to AuNPs changed the surface properties, and the high surface charge of the AuNPs contributes to their excellent partitioning. , Also, as the molecular weight of dextran increases, the gap between polymer networks decreases, reducing the space available for molecules in the dextran phase.…”

“…It is evident that at higher dextran molecular weight, an increase in nanoparticle size has a greater impact. Solute size has been reported to be a key factor in determining the partition coefficient in an ATPS. , However, it is challenging to explain the impact of size due to the lack of control over other variables, such as shape, presence, distribution of charged, hydrophobic, and hydrophilic groups on the biomolecule surface. , In this study, different sized AuNPs/insulin conjugates were used to investigate the effects of size on partitioning. The model demonstrates a significant correlation between AuNPs/insulin partitioning and conjugate size.…”

“…There is a direct correlation between the partitioning of biomolecules, which is quantified by the partition coefficient ( K ), and their physicochemical properties. These properties include isoelectric points, sizes, surface hydrophobicity, polymer molecular weight, polymer/salt concentrations, pH, and temperature. ,, The separations’ selectivity in ATPS has been enhanced under various conditions, which are problematically associated with the need to make structural changes in ATPSs or to modify biomolecules. , It is therefore becoming more necessary to find techniques that do not require the reformulation of system structures or biomolecules. By creating a mechanism that facilitates the transfer of the biomolecule between the two phases, biomolecules partitioning enhances in ATPS. , Nanoparticles fulfill this demand and improve biomolecule partitioning.…”

Plant antimicrobial peptides: a comprehensive review of their classification, production, mode of action, functions, applications, and challenges

“…79 Due to the use of two aqueous phases, ATPS has proved to be largely successful in overcoming the limitations of conventional organic aqueous extraction, including the low solubility of proteins in organic solvents and the tendency of organic solvents to denature them, and for the extraction and purification of biological macromolecules, aqueous two-phase extraction (ATPE) has been found to be a superior and adaptable method. 80…”