Potassium Phosphate System Research Articles

The stabilization mechanism of high-efficiency magnesium phosphate cement for arsenic remediation in lollingite polluted environments

Industrial non-ferrous metal smelting and processing activities are a major contributor to the migration of arsenic-containing minerals throughout the environment, such as lollingite, which poses a severe threat to human health due to its rapid oxidation and subsequent release of arsenic (As). Herein, two phosphate mineral systems (potassium phosphate and calcium phosphate) were used to explore the potential for As solidification and establish the As immobilization mechanisms. Experimental results show that the compressive strength of the calcium phosphate system was higher than that of the potassium phosphate system due to the higher bond energy of the Ca–O–Mg–O–P–O system (31109 kJ /mol), as compared to the K–O–Mg–O–P–O system (16189.2 kJ /mol). Furthermore, the coordination capability of arsenic oxyanions with Ca or Fe ions was stronger than that of phosphorus oxyanions with Ca or Fe ions. The main mechanism of inhibition of As migration by magnesium phosphate cement (MPC) binders, was the formation of FeAsO4.H2O, Ca3(AsO4)2.H2O, MgHAsO4.H2O, CaMgAsO4(OH) and KMgAsO4.6H2O coprecipitates, with physical encapsulation by MgKPO4·6H2O (K-struvite), Ca2Mg(PO4)2·2H2O (Ca-struvite) and Mg2PO4(OH) phosphate-gel. Using Fe2O3-modified MPC binders with a Fe/As molar ratio of 3, the As immobilization efficiency reached 98.73%. The results of toxicity characteristic leaching procedure (TCLP) analysis confirmed that the maximum leached As concentrations from MPC binders cured for 7 days and 28 days were 0.31 mg/L and 0.06 mg/L, respectively, both of which were below the maximum permitted value for safe disposal (5 mg/L). These results verify the potential of MPC binders to be utilized as low-environmental impact and high-efficiency cementitious matrices for As pollution control and environmental remediation.

Partition Efficiency of Eccentric Coil for Countercurrent Chromatographic Separation of Proteins Using Small-scale Cross-axis Coil Planet Centrifuge with Circular and Elliptic Cylindrical Columns.

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

Effect of flow patterns on bovine serum albumin and ampicillin partitioning using aqueous two-phase systems in microdevice

The separation and purification of biomolecules via aqueous two-phase extraction (ATPE) has encountered a series of obstacles to its industrial implementation. Recently, the miniaturization of ATPE has attracted interest due to the increase of performance, the possibility of operating in a continuous mode, and easy to scale-up by numbering-up approach. However, there is a gap in the literature with regard to the effects of flow patterns on the partitioning of biomolecules using microdevices. Thus, the present study investigated the ATPE of bovine serum albumin (BSA) and ampicillin, a beta-lactam antibiotic, under different flow patterns (slug, droplet and parallel) by means of microdevices. The microdevices were manufactured with circular tubes in a senoidal shape and the ATPE were prepared from the mixture between aqueous solutions of polyethylene glycol (PEG) and dibasic potassium phosphate. Using 1.80 mm tubes, slug, droplet and parallel flow patterns were observed only in the PEG 1500/dibasic potassium phosphate system, while the parallel flow pattern was not stabilized in systems involving PEG 4000 and PEG 6000. Experiments using a droplet flow pattern showed higher values of recovery (56% after 10 min) and partition coefficient of BSA compared to the slug and parallel flow patterns. In experiments involving ampicillin, the extraction performance was practically independent of the flow patterns. The BSA recovery behaved as an inverse function of the diameter tubes, which achieved slightly superior performance than the batch experiment (67% after 10 min) using droplet flow pattern, 0.51 mm tubes and residence time of 10 min (69%).

Aqueous two-phase extraction of R-Phycoerythrin from marine macro-algae, Gelidium pusillum

Aqueous two-phase extraction (ATPE) of R-Phycoerythrin (R-PE), a fluorescent and commercially valuable protein, was carried out in two parallel schemes. In scheme-1, ATPE alone was employed, and in scheme-2, process-integration was attempted, where primary extract was subjected to precipitation before standardization of process parameters of ATPE. Scheme-1 with polyethylene glycol (PEG)-3350/potassium phosphate system has resulted in very low R-PE yield (26% w/w) without much enrichment in purity (0.3). In scheme-2, PEG-1450/potassium phosphate system (pH 6, tie-line length 12.26% and lower volume-ratio) has resulted in higher R-PE purity (0.74) with 72% yield. R-PE containing PEG-rich (top) phase was subjected to ultrafiltration, to remove phase forming components, resulting in further increase in R-PE purity (1.1). Overall enrichment in R-PE purity of 11-fold with 57% (w/w) yield and removal of about 95% of total sugars was achieved in scheme-2. Intactness of R-PE after processing was confirmed by absorbance and emission spectrum analysis.

Primary recovery of bioactive compounds from stressed carrot tissue using aqueous two‐phase systems strategies

AbstractBACKGROUNDThe application of controlled postharvest abiotic stresses in carrots induces the production of phenolic compounds, mainly chlorogenic acids, which present diverse biological functions. Aqueous two‐phase systems (ATPS) is a liquid–liquid extraction technique that has demonstrated its potential for the fractionation and recovery of bioactive compounds. ATPS have the advantages of using environmentally friendly constituents, ease of scale‐up and process integration capability. This experimental work addresses for the first time the recovery of bioactive compounds (mainly hydroxycinnamic acid derivatives) from abiotically stressed carrot tissue using ATPS strategies, including in situ extraction, in alcohol–salt and ionic liquid–salt systems.RESULTSThe partitioning of chlorogenic acid was favored in the ethanol–potassium phosphate system achieving a recovery yield of 81.9%. Hydroxycinnamic acid derivatives fractioning was favored in 1‐ethyl‐3‐methylimidazolium acetate (EMIM‐Ac)–potassium phosphate ATPS, where recovery yields up to 85.4% were achieved. The in situ extraction strategy not only demonstrated potential for merging extraction and recovery in the same system but also, depending on ATPS used, allowed differential phenolic profiles to be obtained.CONCLUSIONATPS strategies (either solid–liquid extraction followed by ATPS or in situ extraction) were shown to have great potential for the recovery of bioactives from stressed carrot tissue. © 2014 Society of Chemical Industry

Liquid–Liquid Equilibrium Data for Aqueous Two-Phase Systems Containing PPG725 and Salts at Various pH Values

The phase diagrams and compositions of coexisting phases have been determined for three aqueous two-phase systems (ATPS's) containing poly(propylene glycol) (PPG) with an average molecular weight of 725 and sodium citrate, potassium phosphate, or magnesium sulfate at 25 °C and several pH values. The effect of pH on the salting-out effect of salts in the polymer phase by sodium citrate, potassium phosphate, and magnesium sulfate has been studied. It was found that an increase in salt composition at constant pH increases the concentration of PPG in the top phase and an increase in pH caused the expansion of the two-phase region for these systems. Increasing the pH also increases the concentration of PPG in the PPG-rich phase for systems containing sodium citrate and magnesium sulfate and decreases it for systems containing potassium phosphate, while the salt-rich phase for sodium citrate and magnesium sulfate systems will be more diluted and for potassium phosphate system will be somewhat concentrated. Furt...

Direct Purification of Pectinase from Mango (Mangifera Indica Cv. Chokanan) Peel Using a PEG/Salt-Based Aqueous Two Phase System

An Aqueous Two-Phase System (ATPS) was employed for the first time for the separation and purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel. The effects of different parameters such as molecular weight of the polymer (polyethylene glycol, 2,000–10,000), potassium phosphate composition (12–20%, w/w), system pH (6–9), and addition of different concentrations of neutral salts (0–8%, w/w) on partition behavior of pectinase were investigated. The partition coefficient of the enzyme was decreased by increasing the PEG molecular weight. Additionally, the phase composition showed a significant effect on purification factor and yield of the enzyme. Optimum conditions for purification of pectinase from mango peel were achieved in a 14% PEG 4000-14% potassium phosphate system using 3% (w/w) NaCl addition at pH 7.0. Based on this system, the purification factor of pectinase was increased to 13.2 with a high yield of (97.6%). Thus, this study proves that ATPS can be an inexpensive and effective method for partitioning of pectinase from mango peel.

PARTITION EFFICIENCY OF HIGH-PITCH LOCULAR MULTILAYER COIL FOR COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF PROTEINS USING SMALL-SCALE CROSS-AXIS COIL PLANET CENTRIFUGE AND APPLICATION TO PURIFICATION OF VARIOUS COLLAGENASES WITH AQUEOUS-AQUEOUS POLYMER PHASE SYSTEMS

Partition efficiency of the high-pitch locular multilayer coil was evaluated in countercurrent chromatographic (CCC) separation of proteins with an aqueous-aqueous polymer phase system using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The separation column was specially made by high-pitch (ca 5 cm) winding of 1.0 mm I.D., 2.0 mm O.D. locular tubing compressed at 2 cm intervals with a total capacity of 29.5 mL. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate system (pH 9.2) under 1000 rpm of column revolution. This high-pitch locular tubing yielded substantially higher stationary phase retention than the normal locular tubing for both lower and upper mobile phases. In order to demonstrate the capability of the high-pitch locular tubing, the purification of collagenase from the crude commercial sample was carried out using an aqueous-aqueous polymer phase system. Using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system (pH 6.6), collagenase I, II, IV, V, and X derived from Clostridium histolyticum were separated from other proteins and colored small molecular weight compounds present in the crude commercial sample. Collagenase from C. histolyticum well retained its enzymatic activity in the purified fractions. The overall results demonstrated that the high-pitch locular multilayer coil is effectively used for the CCC purification of bioactive compounds without loss of their enzymatic activities.

Separation of recombinant β-glucuronidase from transgenic tobacco by aqueous two-phase extraction

Transgenic plants hold many promises as viable production hosts for therapeutic recombinant proteins. Many efforts have been devoted to increase the expression level of the proteins, but the efforts for developing economic processes to purify those proteins are lacking. In this report, aqueous two-phase extraction (ATPE) was investigated as an alternative for the separation of an acidic recombinant protein, β-glucuronidase (rGUS), from transgenic tobacco. Screening experiments by fractional factorial designs showed that PEG concentration and ionic strength of the system significantly affected the partitioning of native tobacco proteins and GUS. Response surface methodology was used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4% (w/w) PEG 3400/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while more than 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process. The most important impurity from tobacco, Rubisco, was largely removed from the rGUS in the recovered phase.

Partition Efficiency of Newly Designed Locular Multilayer Coil for Countercurrent Chromatographic Separation of Proteins Using Small-Scale Cross-Axis Coil Planet Centrifuge with Aqueous-Aqueous Polymer Phase Systems

Countercurrent chromatographic performance of the locular multilayer coil separation column newly designed in our laboratory, was evaluated in terms of theoretical plate number, peak resolution, and retention of the stationary phase in protein separation with an aqueous polymer phase system, using the small-scale cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory. The locular column was made from 1.0 mm I.D., 2.0 mm O.D., or 1.5 mm I.D., 2.5 mm O.D. PTFE tubing, compressed with a pair of hemostat at 2 or 4 cm intervals. The protein separation was performed using a set of stable proteins including cytochrome C, myoglobin, and lysozyme with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system under 1000 rpm of column revolution. The 1.5 mm I.D., 2.5 mm O.D. locular tubing compressed at 2 cm intervals yielded better partition efficiencies than the non-clamped tubing, using both lower and upper mobile phases with satisfactory retention of the stationary phase. The overall results suggest that the newly designed locular multilayer coil is useful to the preparative separation of proteins with aqueous-aqueous polymer phase system using our small scale X-axis CPC.

Countercurrent chromatographic separation and purification of various ribonucleases using a small-scale cross-axis coil planet centrifuge with aqueous–aqueous polymer phase systems

Countercurrent chromatographic (CCC) separation and purification of various ribonucleases (RNases) was performed using the small-scale cross-axis coil planet centrifuge (X-axis CPC) with aqueous–aqueous polymer phase systems. RNases B and A were well resolved from each other with an aqueous–aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate (pH 9.2) as the mobile lower phase. The commercial RNase A samples obtained from three different companies were also highly purified using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system (pH 6.6) using the upper phase as the mobile phase. Recombinant RNase Po 1, an RNase T 1 family enzyme, was further successfully separated from the crude extract using the same solvent system with the lower phase used as the mobile phase. The RNase activities were well preserved during the CCC separation. The overall results demonstrate that the small-scale X-axis CPC is useful for a simple and rapid purification of various RNases in a preparative-scale.

Extraction and Purification of C-Phycocyanin from Spirulina Platensis Employing Aqueous Two Phase Systems

Aqueous two phase extraction was employed directly to the cell homogenate of Spirulina platensis for the downstream processing of C-phycocyanin. This enables integrating the process steps of cell removal, extraction and concentration into a single unit operation of aqueous two phase extraction. The effect of different parameters such as molecular weight, tie line length, volume ratio and neutral salt (NaCl) were studied employing PEG 4000/potassium phosphate system and PEG 1000/sodium sulfate system to select the best system among the two. For PEG 4000/potassium phosphate system at 13.12% TLL, and volume ratio 0.45, 90.34% yield of phycocyanin with purity of 0.73 was obtained. In the case of PEG 1000 /Na2SO4 system at 34.98 % TLL and volume ratio of 1, 97.62 % yield of phycocyanin with a purity of 0.41 was achieved. Effect of NaCl concentration shows a significant decrease in K of phycocyanin in PEG 1000/Na2SO4 system as compared to PEG 4000/potassium phosphate system. PEG 4000 and potassium phosphate system was found to be better than PEG 1000 and sodium sulfate system for the extraction and purification of C-phycocyanin.

New small-scale cross-axis coil planet centrifuge: Partition efficiency and application to purification of bullfrog ribonuclease

The new small-scale cross-axis coil planet centrifuge (X-axis CPC) previously designed and fabricated in our laboratory has a distinctive feature such that four separation columns of similar weight are mounted symmetrically around the rotary frame to achieve stable balancing of the centrifuge under a high revolution speed. In this column layout, neighboring columns must be rotated in the opposite direction if viewed from the center of the centrifuge to avoid twisting the interconnecting flow tubes. The effect of rotational direction of the columns on the partition efficiency was evaluated with separation of a set of test samples such as cytochrome c, myoglobin, and lysozyme using an aqueous–aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate under 1000 rpm of column revolution. A series of experiments was performed using a set of two diagonally located columns (connected in series) each consisting of five coiled layers of 1 mm I.D. with a total capacity of 27.0 mL. Both right- and left-handed coils were tested each under the optimized conditions for choice of mobile phase and direction of the column rotation so that the satisfactory volume of the mobile phase was retained in the column by the aid of Archimedean screw effect. The results of these studies showed that one particular combination of handedness of the coil and direction of the rotation yielded the best peak resolution for each mobile phase. In order to demonstrate the capability of the apparatus, the purification of ribonuclease (RNase) from the extract of bullfrog egg, sialic acid binding lectin (cSBL), was carried out using both organic–aqueous and aqueous–aqueous polymer phase systems. When using the 16.0% (w/w) PEG 1000–6.3% (w/w) dibasic potassium phosphate–6.3% (w/w) monobasic potassium phosphate system, cSBL was successfully separated from other proteins present in the extract while commercial RNase A was eluted at near the solvent front by the lower phase mobile. The cSBL retained its native RNase activity. The overall results demonstrated that the present new small-scale X-axis CPC is useful for the purification of bioactive compounds without loss of their native activities.

Aqueous two phase extraction for purification of C-phycocyanin

Aqueous two phase extraction is employed for the purification of C-phycocyanin from Spirulina platensis. A systematic approach is suggested to arrive at the optimal process parameters of aqueous two phase extraction by considering a case study of C-phycocyanin. The influence of various process parameters such as type of aqueous two phase systems, phase forming salt, molecular weight of the phase forming polymer, system pH, phase composition, phase volume ratio, and type and concentration of neutral salts on differential partitioning of C-phycocyanin is evaluated. Desirable conditions for the purification of C-phycocyanin are found in polymer–salt systems especially in polyethylene glycol (4000)/potassium phosphate system. Increase in purity of C-phycocyanin to 3.52 from initial purity of 1.18 is achieved at pH 6, tie line length of 35.53% with a phase volume ratio of 0.8 in a single step of aqueous two phase extraction. Multiple extractions resulted in further increase in purity of C-phycocyanin without loosing the yield and a maximum purity of 4.05 is achieved in third aqueous two phase extraction. The integration of ultrafiltration with aqueous two phase extraction facilitated the selective removal of polyethylene glycol from the purified C-phycocyanin. Finally, C-phycocyanin is freeze dried to obtain in powder form.

Purification of alcohol dehydrogenase from bovine liver crude extract by dye–ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye–ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge ( x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000–7.0% dextran T500–0.1 M potassium phosphate buffers and 16% PEG 1000–12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000–potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6 h with minimum loss of its enzymatic activity.

Protein separation by nonsynchronous coil planet centrifuge with aqueous–aqueous polymer phase systems

Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous–aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000–dibasic potassium phosphate, and PEG 8000–dextran T500 in 5 m M potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000–12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous–aqueous polymer phase systems.

Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate.

Purification of recombinant wild-type cutinase from the culture supernatant of Saccharomyces cerevisiae by extraction in aqueous two-phase system was investigated. The partition of the enzyme in a polyethylene glycol (PEG)-potassium phosphate system to the top phase was increased with lower molecular weight PEG. Enzyme partition in a 20% PEG/15% phosphate two-phase system was studied in the presence of detergents, fatty acids, and alcohols, respectively. Addition of 0.5% (w/w) butyrate increased the partition coefficient from 17 to 135 and the purification factor from 10 to 23. The effect of butyrate was also confirmed by using the countercurrent mode of extraction. Recovery of cutinase from the top phase was achieved by a secondary extraction into a new salt phase at a lower pH or a lower temperature. A specific interaction of butyrate to the active site of the enzyme was demonstrated by fluorescence spectroscopy. Size exclusion chromatography showed the cutinase-butyrate complex to be over two times the size of the free enzyme.

Countercurrent Chromatographic Separation of Proteins by Cross-Axis Coil Planet Centrifuge: Choice of Polymer Phase Systems and Revolution Speed

Countercurrent chromatographic separation of proteins by the cross-axis coil planet centrifuge was maximized by selecting the suitable polymer phase system and revolution speed. Polymer phase systems composed of polyethylene glycol (PEG) 1000 and several inorganic salts were examined to determine partition coefficient values (K) for various proteins. The overall results indicated that the polymer phase system composed of 12.5% (w/w) PEG 1000 and 12.5% (w/w) dibasic potassium phosphate yielded suitable K values for most proteins except for cytochrome C and apo-transferrin which may be separable with a solvent system composed of 12.5% (w/w) PEG 1000 and 24% (w/w) potassium citrate. A series of experiments with the PEG 1000 - potassium phosphate system under various revolution speeds revealed that the best separation was achieved at 850 rpm. The above optimized conditions may be applied to separations of other protein samples.

Use of aqueous two-phase systems in the purification of human interferon-α1 from recombinant Escherichia coli

Intracellular human recombinant interferon α1 (rhIFN-α1) has been purified using two consecutive aqueous two-phase extractions: selective extraction followed by back extraction. In the selective extraction step using polyethylene glycol (PEG)-potassium phosphate aqueous two-phase systems, optimization of the systems was achieved by varying the concentrations of PEG-phosphate ester, phosphate, added inorganic salts, pH, and biomass load. The optimum condition was found to be 22% (w/w) synthesized PEG-phosphate ester, 16% (w/w) potassium phosphate and 3% (w/w) NaCl at pH 6.9 with 10% (wet w/w) biomass load. The corresponding top phase yield of rhIFN-α1 was 99.6% with a purification factor of 25. It was observed that most of the modified PEG is partitioned to the phosphate-enriched bottom phase, so the mechanism for enhancement to the extraction is likely to be an electrostatic expulsion effect. In the back extraction step where the partially purified IFN is transferred from the PEG-rich top phase to a phosphate buffer, the concentrations of PEG-phosphate ester and phosphate, pH, and other parameters have been adjusted. The pH was found to be the most sensitive. A back extraction system using 20% (w/w) PEG-phosphate ester and 10% (w/w) potassium phosphate system at pH 6.0 gives the yield of rhIFN-α1 in the bottom phase (approximately 76%). This newly established back extraction procedure avoids the use of a cumbersome gel chromatography while the yield achieved is fairly high.

Predicting partition coefficients of multi-charged solutes in aqueous two-phase systems

A mathematical model incorporating the influence of a pH difference between the phases is developed to predict the partitioning of charged compounds in aqueous two-phase systems. The partition coefficients of three model compounds, terephthalic acid, benzenedimethanol and xylylenediamine, are examined in poly(ethylene glycol)-potassium phosphate aqueous two-phase systems over the pH range 5.5–9.2. The model predicts that in this particular phase system, negatively charged solutes partition higher and positively charged solutes partition lower than an otherwise identical neutral solute. The partition coefficients of the dipeptides tyrosine-tyrosine, glycine-tyrosine and leucine-phenylalanine are also examined in poly(ethylene glycol)-potassium phosphate systems, and their observed behaviour agrees with the model prediction. The addition of an alkali metal halide to a poly(ethylene glycol)-potassium phosphate system was observed to result in a decrease in the pH difference between the phases. As the model predicts for decreasing pH difference between the phases, the observed partition coefficients of negatively charged compounds decrease. The results indicate that charge and hydrophobic effects each play important roles in the partitioning of biological compounds.