Affinity partitioning systems

Johansson, G. (1992) Affinity partitioning in PEG-containing two-phase systems. In Poly(Ethylene Glycol) Chemistry Biotechnical and Biomedical Applications (J.M. Harris, ed.), pp. 73-84. Plenum, New York. 

Other applications besides purification of biomolecules are affinity partitioning (Labrou, 1994), extractive bioconversions (Andersson, 1990 Kaul, 1991 Zijlstra, 1998), liquid-liquid partition chromatography, and analytical assays. Although industrial applications of aqueous two-phase systems have not gained widespread use to date, practical and economic feasibility has been proved (Tjemeld, 1990 Cunha, 2002). 

Johansson G (1998), Affinity partitioning of proteins using aqueous two-phase systems, In Janson JC, Ryden L (Eds), Protein Purification Principles, High-Resolution Methods, and Applications, 2nd Ed., Wiley-VCH, Weinheim, pp. 443-460. 

TABLE 11 Affinity Partitioning of Biomolecules in Aqueous Two-Phase Systems... 

Harris, J. M., and Yalpani, M. (1985). Polymer-ligands used in affinity partitioning and their synthesis. In Partitioning in Aqueous Two-Phase Systems Theory, Methods, Uses, and Applications to Biotechnology (H. Walter, D. E. Brooks, and D. Fisher, eds.), pp. 589-626. Academic Press, New York. 

These measurements were conducted in ATP system consisting of 5.1% (w/w) PEG 8000 and 10% (w/w) dextran 70 in phosphate buffer at 4°C. Under these conditions, the volume ratio of PEG (light) to dextran (heavy) phases was 3 7. The composition (w/w) of PEG phase was 9.25% PEG and 0.52% dextran. The composition of the dextran phase was 0.54% PEG and 21.1% dextran. The phase composition was unaffected by NaCl concentration in the range of 0-5 M. The partition coefficient is defined as ratio of concentration in PEG phase to that in dextran phase. The triazine dye, Cibacron blue FGF, was ligated to PEG for affinity partitioning. 

Mattiasson, B. Ling, T.G.I. Efforts to integrate affinity interactions with conventional separation technologies affinity partition using biospecific chromatographic particles in aqueous two-phase systems. J. Chromatogr. 1986, 376, 235-243. 

Kopperschlager, G. Lorenz, G. Usbeck, E. "Application of Affinity Partitioning in an Aqueous Two-Phase System to the Investigation of Triazine Dye-Enzyme Interactions" /. Chromatography. 1983, 259, pp 97-105. 

Firary, M. Carlson, A. "Affinity partitioning of Acid Proteases in the Hydrox)q)ropyldextran-dextran Aqueous Two-Phase System" presented at the AIChE Summer Meeting, Boston, M A, August 1986. 

The main drawback to the widespread use of polymer-polymer aqueous two-phase extraction has been the high cost of fractionated dextran. Crude dextran has been used with some success for the purification of enzymes but is much too viscous for many applications. Conversely, polymer-salt systems have relatively low viscosities, separate rapidly, and are inexpensive. Unfortunately, they lack selectivity and cannot be used for affinity partitioning in most cases since the high salt concentrations interfere with the protein-ligand interaction. The starch derivatives are reasonable alternatives for bottom phase polymers but have been hampered by low solubilities and the tendency for gel formation. Tjemeld has reported that chemically modified starches i.e. hydroxypropyl starch... 

The MD/PEG system offers the combined advantages of low-cost, reduced lower phase viscosities and high density differences for inexpensive polymer-polymer affinity partitioning. When coupled with low-cost affinity ligands i.e. triazine dyes, two-phase aqueous affinity partitioning could be used as the first step in a separation train for the recovery of industrially important enzymes. The bottom phase, which is generally considered to be a waste stream and non-recyclable if dextran or salt is used could be used as a substrate for additional fermentations if maltodextrin is used, thereby aiding the overall economics of the process. 

Affinity partitioning. In some cases, it may not be possible to separate one protein out of a complex mixture by means of spontaneous partitioning in an aqueous two-phase system. Then affinity interactions may be utilized. When first described, affinity partitioning was used for purification of membrane vesicles (26), and has since been exploited in a broad spectrum of applications. 

Table III. Examples of affinity partitioning in aqueous two-phase systems.

Extraction in aqueous two-phase systems is said to be an operation that is easy to scale up (33). This is also true for affinity partitioning and is clearly illustrated by the process for purification of formate dehydrogenase (34, 35). In this study, small scale experiments gave an overall enzyme yield of 74%. When scaled up by a factor of 40 000, the yield was 70%, thus demonstrating the feasibility of evaluating the performance of extraction process on a small scale. 

A reason why the PEG/salt systems have not been used more extensively is that the high ionic strength severely influences the affinity interactions when affinity partitioning is to be exploited. However, in applications where spontaneous partitioning is sufficient, these systems are often used. Recently, the use of starch derivatives named as Reppal PES has been reported (32) that are useful in forming aqueous two-phase systems. The properties of these new polymers resemble those of dextran. 

Avidin Chicken egg-vsfhite Activated PEGa Affinity partitioning Aqueous two-phase system 118... 

Affinity values aie obtained by substituting concentiation foi activity in equation 4 foi the dye and, wheie appropriate, other ions in the system. A number of equations are used depending on the dye—fiber combination (6). An alternative term used is the substantivity ratio which is simply the partition between the concentration of dye in the fiber and dyebath phases. The values obtained are specific to a particular dye—fiber combination, are insensitive to hquor ratios, but sensitive to all other dyebath variables. If these limitations are understood, substantivity ratios are a useful measure of dyeing characteristics under specific appHcation conditions. 

Liquid-liquid extraction is a basic process already applied as a large-scale method. Usually, it does not require highly sophisticated devices, being very attractive for the preparative-scale separation of enantiomers. In this case, a chiral selector must be added to one of the liquid phases. This principle is common to some of the separation techniques described previously, such as CCC, CPC or supported-liquid membranes. In all of these, partition of the enantiomers of a mixture takes place thanks to their different affinity for the chiral additive in a given system of solvents. 

In some metal components it is possible to form oxides and carbides, and in others, especially those with a relatively wide solid solubility range, to partition the impurity between the solid and the liquid metal to provide an equilibrium distribution of impurities around the circuit. Typical examples of how thermodynamic affinities affect corrosion processes are seen in the way oxygen affects the corrosion behaviour of stainless steels in sodium and lithium environments. In sodium systems oxygen has a pronounced effect on corrosion behaviour whereas in liquid lithium it appears to have less of an effect compared with other impurities such as C and Nj. According to Casteels Li can also penetrate the surface of steels, react with interstitials to form low density compounds which then deform the surface by bulging. For further details see non-metal transfer. 

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