Partitioning in Aqueous Two-Phase System. Theory, Methods, Uses, And Applications To Biotechnology

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Li and H. Sthrathmann eds. Tjerneld, F. Enzyme Microb. Szlag, D. Techniques 2 : Hamel, J. Hunter, and S. Sikdar eds. Giuliano, K. Unpublished research. Wuenschell, G. Stewart, R. Sikdar, M. Bier, and P. Todd eds. Cole, K. BioTechniques , in press. Abstract No. Cabezas, Jr. Hill, T. Theory of Solutions II. Fluid Phase Equilibria. Kabiri-Badr, M. Forcinit, D. King, R. AIChE J. Kang, C. Macromolecules 21 : — Joshi, J. Bioseparation , in press. Raghava Rao, K. Raghave Rao, K. Electrokinetic Demixing of Immiscible Aqueous Solutions.

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Partitioning Between Liquid Phases

Log In Sign Up. Ana Azevedo. Raquel Aires-Barros, and Ana M. After many years with only a few articles published, a lot of effort and work has been put into studying these systems for the par- titioning of a range of compounds including proteins, organic low-molecular weight molecules or metal ions. Although several research and review articles appeared, a background review on ATPS partitioning fundamentals is needed.

In this article, partitioning theories and main effects of several important factors for partitioning, such as molecular weight of the polymer, effect of added salts, pH, electrical charges, and temperature on phase diagrams, tie-line lengths, interfacial tension and settling time of the two aqueous phases are extensively reviewed.

The trend in ATPS research is given compiling the recent — research articles published in the field. As for many other relevant events in science and technol- ATPSs present an opportunity for the application of ogy, such incompatibility was found by accident and it took liquid-liquid extraction concepts from classical chemical many years until an application was found for these sys- engineering to the purification of biological products. The tems. Professor Albertsson inspired a generation biocompatible components polymers and salts in a gen- of scientists and engineers who have been working on the tle aqueous environment.

It has been reported that, for the applications of ATPSs in several fields leading to hundreds purification of biomolecules, the most important features of ATPSs are low interfacial tension and high water con- tent which preserves biological molecules activity 5, 6. Gu Received 17 March , Accepted 27 October Azevedo, IBB - Institute for feron and glucosephosphate dehydrogenase, and cellular Bioengineering and Biosciences, Department of Bioengineering, Instituto components that have been purified at large scale 0.

Rovisco Pais, Lisbon , Portugal. E-mail: a. ATPSs were This collapsing phenomenon can be driven by temper- also employed to extract biological products from fermenta- ature, pH, ionic strength or light. Thermosensitive polymers, tion broths 18, A significant advantage of ATPS is the have been widely used for aqueous-two-phase partitioning simplicity of the required equipment These one-component ATPSs are very interesting as Over the last — years, about research articles they allow for easy polymer recycling.

Azevedo et al. The antibodies were extracted in the cold poly- mer phase. Upon heating above the polymer cloud-point, Downloaded by [b-on: Biblioteca do conhecimento online UTL] at 13 November ATPS can be formed with a variety of components two phases formed and the mABs were collected into the in aqueous solution. Two-polymer systems, especially water phase. However, poly propylene glycol PPG 52 , poly ethylene oxide sul- it did not take long until dextran alternatives started to fide PEOS 60 , biopolymers 61 , alcohols 62, 63 , be used 27— Dextran is an expensive polymer com- organic acids 64 , surfactants 55, 65—67 or, more recently, pared to PEG alternatives—mostly thermoseparating ethy- ionic liquids 42, 68— Citrate 34—40 , phosphate 26, 41—49 in different phase forming agents.

A statistical study based and sulfate 38, 50—57 are the most commonly used salts. Accordingly, water one-component ATPS. One article may describe more than one type of system. Upper left insert: number of ATPS-focused research articles published over the last 6 years — Hydrophobic affinity — separation occurs according to Albertsson in When a stable two-phase system forms, hydrophobic interactions between the molecules and Downloaded by [b-on: Biblioteca do conhecimento online UTL] at 13 November its two co-existing phases are at equilibrium.

The chemical potential can be specific binding sites for the target molecule; expressed as a function of activity ai , particle surface area 5. At equilibrium, Eq. Then Eq. KZ depends on the net charge of the protein pH depending and its interaction with the two phases which At infinite dilution, the activity coefficients approach ultimately depend on ionic strength 4.

Then, Eq. Bolognese et al. The model derived by Albertsson shows that the per- Cabezas reviewed more than five different thermody- formance of an ATPS is affected by several factors Among the examined meth- tors are simply by analyzing Eq. Indeed, the mechanisms ods, the osmotic-virial equations seem to provide very good that describe partitioning are, so far, poorly understood 14, results 76 and have also been investigated by King et al.

Their model provides a rather detailed descrip- Two-Liquid NRTL model that can accurately predict a Downloaded by [b-on: Biblioteca do conhecimento online UTL] at 13 November tion of the interactions between the solute and the polymer series of the liquid-liquid equilibrium phase diagrams of aqueous solutions but does not account for the effects of ATPS at different polymer molecular weights In , pH or salts The model relies on five questionable Perez et al.

On one side there are authors such as Luechau et al. Coefficient A accounts for i polymer and protein molec- supporting the excluded volume theory. He described par- ular weights, ii protein charge, iii protein interactions with titioning of nucleic acids and concluded that the excluded- water, polymers and other protein molecules, iv pH, v elec- volume theory explained well all experimental results Coefficient b accounts for protein hydrophobic interactions provide the most relevant expla- molecular weight and charge, polymer-water interactions nation for ATPS partitioning The model showed good agreement with the exper- high molecular-weight PEGs.

For lower molecular weights, imental results for most studied proteins, however, due to the authors raise the hypothesis of PEG-protein complexes mathematical limitations, it is only valid for intermediate based on unknown affinity interactions Noteworthy, ranges of polymer concentration differences between the there are also scientists who believe electrostatic interactions phases Indeed, as the critical point is approached, top dominate partitioning.

However and description of affinity partitioning Madeira et al. Kp Model used Model Kp Ref. BSA 8. Factors Influencing ATPS Partitioning The TLL is also a measurement of how broad the two- phase region in the phase diagram is or, in other words, how As it is not yet possible to describe partitioning mecha- incompatible the two phase-forming components are. For nisms in ATPS, most experimental work has been relying analytical applications, the phase diagram and, in particular, on product-based system optimization.

In this context, a the TLL is important in order to obtain partitioning into the wide range of factors have been studied. Johansson stud- desired phase at the lowest possible phase volume. The goal ied a range of properties of two different aqueous two-phase is to concentrate the product as much as possible in order systems for the partitioning of proteins and concluded that to achieve cost reduction and to apply standard analytical the tie-line length TLL is a good parameter to account for methods that typically require dilution of the phases.

The authors showed are now overviewed 20, Higher molecular weight polymers form two phases Polymer Concentration and Molecular Weight at lower concentrations compared to similar lower MW All ATPS include at least one polymer and it is there- polymers. Tjerneld and Albertsson also report dissymmetric fore important for the study of ATPS to understand how binodal curve as the difference between the molecular weight polymer solutions behave in different conditions.

Regarding of the phase-forming polymers increases in a two-polymer polymer concentration in water, two possible regimes may ATPS 5. The former is characterized by having sev- related to partitioning. According to this author, larger pro- eral layers of solvent water in this case keeping polymer teins are more influenced by the molecular weight of the molecules apart.

As the molecular weight increases, the con- polymer which presents an opportunity for size-based par- centration range within which this regime applies becomes titioning The concentrated regime is then used relation — where a is a polymer concentration dependent to describe these solutions. The concentrated regime is ade- constant and M is the protein molecular weight — was quate when the volume occupied by polymer molecules is established by Albertsson 7.

Cabezas used data for PEG and a model to demonstrate how Several ligands can be used to modify polymers to this concentration is related to the molecular weight of the improve partitioning.

Partitioning In Aqueous Two – Phase System ebook |

Sometimes, the use of charged polymer — higher molecular weights correspond to lower polymers is also an alternative Although this affin- crossover concentrations and vice-versa. Although the author highlights fatty acids and thiazine, in ATPS formation. Of course, as the polymer concentration ligands include organic groups, proteins, metals and amino increases, the properties of the polymer solution approach acid tails, as well 7. Affinity partitioning can be accomplished either polymers behave in a dilute regime at higher weight fractions using functionalized polymers or ligands with affinity to both in solution than high-MW ones.

This is exactly the impact the polymer and the target of interest — so called dual-ligand described by Albertsson and Tjerneld in terms of ATPS per- systems. One example of a dual-ligand system is a protein formance 5. The authors established that heavy polymers called Lytag His approximate model Eq. In Eq. Although qualitative, this model provides a of the polymers is necessarily more hydrophobic than the good overall perspective of the partition coefficient variation other. For PEG polymers, for example, as the molecular with the target molecular weight.

In particular, the authors explain the influence Johansson was one of the first to apply the theoretical model of NaClO4. BSA has an isoelectric point pI of 5. When of Albertsson regarding KZ 94 and the same theoreti- the bulk pH is higher than 5. BSA will prefer the phosphate- K- their respective partition coefficients assuming that each rich lower phase thus ensuring charge balance. On a similar ion could partition independently from the others. It is ATPS. It is related also to sulfate was enough to achieve better yields and partition adsorption at the interface.

Typically, the interfacial tension coefficients A large interfacial tension gives an easy phase sep- solute towards either of the two phases of an ATPS leads aration but a difficult dispersion of one liquid into the other, to the establishment of an electric double-layer responsi- hence a difficult solute extraction.

In , Bamberger et al. They further validated their method by using mM 7. Salts have also been reported to affect the cloud influence of dextran molecular weight and temperature. If the point of polymers. This salting-out effect can be used to effect of dextran MW is simple to describe — interfacial ten- lower the cloud point thus allowing for handling biological sion increases with increasing MW — that is not the case with products at lower temperatures 95— When systems with the same com- pH has also an effect on the electrochemical contribution positions are prepared at different temperatures, interfacial to the overall partition coefficient.

By changing the charge tensions increase as temperature decreases. However, if sys- of ionizable groups in molecules, and, possibly, their sur- tems with the same phase separations behavior, i. A good example of the effect of salts and pH on the parti- The Bamberger model was applied to provide an exten- tion of proteins is described by Bolognese and co-workers sive description of the influence of temperature and polymer This MW influence very significant as the critical point is Asenjo and co-workers have also analyzed the relation- approached — i.

The systems and TLL. Their experiments showed that the sepa- interfacial tension changes with temperature, either increas- ration rate is strongly dependent on the viscosity of the top ing or decreasing depending on the TLL, that is, depending PEG phase and on the density of the bottom salt phase Experimental data from this study was used by a research group that worked on modeling of ATPS interfacial Settling Time tension In this work, Wu and Zhu used the NRTL ther- Another very important factor to consider is the separation Downloaded by [b-on: Biblioteca do conhecimento online UTL] at 13 November modynamic model, the concept of interface phase and the role of the electrochemical potential to accurately model the time for phase settling.

Asenjo et al. In the course of their study, the authors state that rate of phase separation and correlated interface tension and phase separation settling time is a function of density and TLL They confirmed that longer TLL corresponded to viscosity of the phases, diameter of the droplets and inter- higher interface tensions.

1st Edition

Similar results were found by Ding face tension. Although phase settling time is very much a low et al. Barnea and Mizrahi published At lab scale, phase settling is often quickly achieved by a separation profile showing that separation is concluded centrifugation thus decreasing separation time from several when the settling of the bottom phase and coalescence of hours to a few minutes. TLL increases is correlated with higher density differ- ences between top and bottom phases and the viscosity of the Although the mechanisms that govern partitioning in ATPSs top PEG phase As density and processing technique and as an analytical tool.

After considering all articles, applications were that they increase with TLL, i. It is inter- is getting deeper into the two-phase region. However, one should the articles. It would therefore be expected that applications- never forget the general rule in upstream-downstream pro- based papers would start emerging. With industry being cessing relationship — everything that goes in, should come increasingly more open to novelty — giving ATPSs a chance out again.

Therefore, if an ATPS is to be used for a spe- — this may happen in the next coming years. So far, this is cific purification, especially the purification of a biological not the case. Applications include concentration eficial contribution to the overall process. Rito-Palomares and quantification of biological solutes as well as label- and Cueto studied the influence of four biological feedstocks free cell analysis. The purification of organic compounds and found that a mycelia culture of T. Interestingly, ATPSs have even been used as reaction curve towards the origin.

Aqueous two-phase system (ATPS): an overview and advances in its applications

This change in the phase diagram medium for organic syntheses. A strong effort is put into studying the systems by themselves and proteins account for the vast majority of applications. In proportion, mAbs account for a small fraction of all works published but their market is the largest. It is also interesting to see that analytical applications are against trend. ISI-indexed articles only, excluding review articles.


One article may describe more than one application. One of the most inter- In , Ahmad and Przybycien have used technical and esting papers was that of Lee and Forciniti where they ana- economic arguments in order to develop an optimized flow lyzed the importance of the glycosylation on the partitioning sheet for ATPS use for protein purification and Espitia- of mABs to conclude that the presence or absence of polysac- Saloma et al. The same group developed a novel forces affect the system The analytical features of ATPSs are also being mixed-settlers battery is used to perform in-series extraction extended to label-free cell separation systems.

High through- steps of mABs from two-different cell culture supernatants. ATPSs have development of one-polymer and polymer-salt APTS for been described for separation and purification of several the harvesting of cells in the downstream processing of types of particles and molecules. In particular, studies on mABs Increasing titers A drawback of ATPSs is the poor understanding and abil- and solids concentrations as a result of more than 20 years ity to model and optimize partition processes.

Sorting out of work on upstream processes improvements are challeng- the mechanisms that rule the partitioning of biomolecules in ing industrial scale downstream processing. These remark- ATPSs will be a major breakthrough. This would definitely able achievements bring the need for alternative harvesting allow for the rational design of ATPSs, including the choice techniques and alternatives ways to established modes of of the phase forming components and separation conditions.

In both cases, aqueous two-phase systems The development of reliable models would predict the par- have shown to have a role of their own in the next generation titioning of a key product from a crude feedstock opening of biological processes. In the several very interesting Downloaded by [b-on: Biblioteca do conhecimento online UTL] at 13 November one and may be helpful to take conclusions for protein applications-based reviews evaluated, the authors have iden- purification processes in general.

Although protein-A affin- tified a lack of fundamental and background information- ity chromatography will hardly be replaced by ATPSs, based works. This compilation of theoretical background current harvesting bottlenecks may be overcome by using articles published so far aims at being a tool for those study- this technique. It provides a ATPSs and they may be implemented at process scale thorough review on the fundamentals as well as an extensive to remove cells together with some impurities.

ATPSs list of references for further research and investigation. Even if ligands are developed to be the favorite purified products and university-industry allowing an ATPS to achieve the performance of a chro- collaborations have shown tremendous synergistic results matographic separation and at the same cost, there is no and hopefully more will be established.