Correlations between distribution coefficients of various biomolecules in different polymer/polymer aqueous two-phase systems

Distribution coefficients for a variety of proteins and certain other biomolecules (peptides, amino acids, and carbohydrates) (overall 27 different solutes) were measured in aqueous two-phase systems (ATPSs) dextran (Dex)–polyethylene glycol (PEG) and Dex–Ucon 50-HB-5100 (Ucon—a random copolymer of ethylene glycol and propylene glycol) both containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4, at 23 °C. Distribution coefficients of some selected solutes were also measured in the above two-phase systems at three different polymer concentrations for each system. It was established that the distribution coefficients for all the proteins examined in the ATPSs are correlated according to the so-called Collander linear equation.     

Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies

The partition of human antibodies in aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and phosphate was systematically studied using first pure proteins systems and then an artificial mixture of proteins containing 1mg/ml human immunoglobulin G (IgG), 10mg/ml serum albumin and 2mg/ml myoglobin. Preliminary results obtained using pure proteins systems indicated that the PEG molecular weight and concentration, the pH value and the salts concentration had a pronounced effect on the partitioning behaviour of all proteins. For high ionic strengths and pH values higher than the isoelectric point (pI) of the contaminant proteins, IgG could be selectively recovered on the top phase. According to these results, a face centred composite design was performed in order to optimise the purification of IgG from the mixture of proteins. The optimal conditions for the isolation of IgG were observed for high concentrations of NaCl and low concentrations of both phase forming components. The best purification was achieved using an ATPS containing 8% (w/w) PEG 3350, 10% (w/w) phosphate pH 6 and 15% (w/w) NaCl. A recovery yield of 101+/-7%, a purity of 99+/-0% and a yield of native IgG of 97+/-4% were obtained. Back extraction studies of IgG to a new phosphate phase were performed and higher yields were obtained using 10% phosphate buffer at pH 6. The total extraction yield was 76% and the purity 100%.     

Evaluation of a PEG/hydroxypropyl starch aqueous two‐phase system for the separation of monoclonal antibodies from cell culture supernatant

In this study, an aqueous two‐phase system (ATPS) with PEG and hydroxypropyl starch (HPS) was used to separate monoclonal antibody (mAb) from Chinese hamster ovary cell culture supernatant. The phase diagram of the PEG/HPS ATPS was determined, and the effects of NaCl addition were investigated. The results showed that NaCl addition could lead to a shift of the binodal curve and that phase separation would occur at higher PEG and HPS concentrations. The effects of NaCl addition, pH, and the load of cell supernatant on the partitioning of mAb in a PEG/HPS ATPS were investigated. It was found that with 6% cell supernatant and 15% NaCl addition at pH 6.0, the yield of mAb in the upper phase was 96.7% with a purity of 96.0%. The back‐extraction of mAb with a PEG/phosphate ATPS were also studied, and the results showed that after the two‐step extraction with ATPSs the purity of mAb could reach 97.6 ± 0.5% with a yield of 86.8 ± 1.0%, which was comparable to the purification with Protein A chromatography. These results indicate that the two‐step extraction with PEG/HPS and PEG/phosphate ATPSs might be a promising alternative for the separation of mAb from cell culture supernatant.     

氨基酸在乙醇/磷酸氢二钾双水相体系中分配行为

研究了5种结构和性质各异的氨基酸(谷氨酸,半胱氨酸,苯丙氨酸,赖氨酸,精氨酸)在亲水性有机溶剂乙醇和磷酸盐溶液形成的乙醇/K2HPO4双水相体系中的分配行为及其影响因素。当双水相体系中乙醇的质量分数从22%增加到30%时,半胱氨酸和苯丙氨酸的分配系数明显增大,其它三种氨基酸的分配系数变化幅度不大;当磷酸盐的质量分数从14%增加到22%时,氨基酸的分配系数的变化均不明显;双水相体系的pH值对氨基酸的分配系数影响较大,当pH增加时,5种氨基酸的分配系数均增加,但是当pH约为9.5时,苯丙氨酸、半胱氨酸的分配系数接近相等但与其它3种氨基酸的分配系数相差较多,当pH为6.0~8.0时,苯丙氨酸的分配系数远远大于另外4种氨基酸。氨基酸的支链结构中含有非极性基团将导致其分配系数的增加。     

Horseradish peroxidase extraction and purification by aqueous two-phase partition

The effect of poly(ethyleneglycol) (PEG) molecular weight, system pH, and sodium chloride concentration on the partitioning behavior of horseradish peroxidase fromArmomcia rusticana root extract was investigated in poly(ethyleneglycol)/sodium phosphate systems. PEG molecular weight strongly affects the enzyme partition coefficient, whereas pH variation from 5.5 to 8.0 has little effect. The addition of sodium chloride (8% w/w) to a PEG 1540/phosphate system, pH 7.0, raises the peroxidase partition coefficient 13.5-fold without important changes in that of total horseradish root proteins. Moreover, these conditions allow direct homogenization of theA. rusticana roots with the selected aqueous two-phase system with the clear top phase containing over 90% of the enzyme and the purification factor being 4.8.     

Xylanase recovery effect of extraction conditions on the aqueous two-phase system using experimental design

The partitioning of xylanase produced byPenicillium janthinellum in aqueous two-phase systems (ATPS) using poly(ethylene glycol) (PEG) and phosphate (K₂HPO₄/KH₂PO₄) was studied employing a statistical experimental design. The aim was to identify the key factors governing xylanase partitioning. The interactions of five factors (PEG concentration molecular weight, concentration of buffer K₂HPO₄/KH₂PO₄, pH, and NaCl concentration) and their main effects on the partition coefficient (K) were evaluated by means of a 2⁵ full-factorial experimental design with four center points. The %PEG, %NaCl, and pH were the most important factors affecting the response variable (K). Response surface methodology (RSM) was adopted and an empirical second-order polynomial model was constructed on the basis of the results. The optimum partition conditions were pH 7.0, PEG = 8.83% and NaCl = 6.02%. Adequacy of the model for predicting optimum response value was tested under these conditions. The experimental xylanase partition coefficient (K) was 2.21, whereas its value predicted by the model was 2.33. These results indicate that the predicted model was adequate for the process. PEG molecular weight and phosphate concentration did not affect the xylanase partition coefficient.     

Effect of anionic structure on the phase formation and hydrophobicity of amino acid ionic liquids aqueous two-phase systems

Compared with the conventional ionic liquids, amino acid ionic liquids are more biodegradable and biocompatible, and can enhance stability of biomaterials. In this work, amino acid ionic liquids 1-butyl-3-methylimidazolium L-serine ([C(4)mim][Ser]), 1-butyl-3-methylimidazolium glycine ([C(4)mim][Gly]), 1-butyl-3-methylimidazolium L-alanine ([C(4)mim][Ala]) and 1-butyl-3-methylimidazolium L-leucine ([C(4)mim][Leu]) have been synthesized. These ionic liquids are found to form aqueous two-phase systems (ATPSs) by the salted-out of K(3)PO(4) in aqueous solutions. Phase diagram of the ATPSs and the Gibbs energies of transfer of methylene group from the bottom salt-rich phase to the top ionic liquid-rich phase have been determined at 298.15K and pH 14, and the effect of anionic structure of the ionic liquids on phase formation of the ATPSs and the relative hydrophobicity between the top and the bottom phases are then examined. In order to understand the effect of relative hydrophobicity of the phases in equilibrium in the ATPSs on the extraction/separation capability of biomolecules, the partition coefficients of cytochrome-c (as a model biomolecule) in the ATPSs are measured by spectrophotometry. It is suggested that hydrophobic interactions are mainly responsible for the higher partition coefficients of cytochrome-c in aqueous two-phase systems at pH 14, and the extraction and separation capacity of biomolecules can be improved by the modulation of the relative hydrophobicity of the phases and/or the pH of the system.     

Solvent systems for countercurrent chromatography: an aqueous two phase liquid system based on a room temperature ionic liquid

A new aqueous two phase liquid system (ATPS) based on the ionic liquid 1-butyl-3-methyl imidazolium chloride (BMIM Cl), potassium dibasic phosphate (K(2)HPO(4)) and water was recently proposed in the literature. The full phase diagram of this ATPS was prepared and some tie lines were fully determined. It was compared to classical ATPSs based on polyethylene glycol with an average molecular mass of 1000 (PEG 1000) and 10,000 (PEG 10000) and K(2)HPO(4). Two countercurrent chromatography (CCC) columns, a hydrostatic Sanki and a J type hydrodynamic CCC columns were used to test the liquid phase retention of these ATPSs in all possible configurations. It was found that the BMIM Cl ATPS liquid phases were much easier to retain in the two CCC columns than the PEG 1000 ATPS phases. Using protein and alcohol solutes, it was established that the BMIM Cl ATPS has a polarity completely different from that of the PEG 1000 ATPS. For example, ovalbumin partitions equally between the two phases of the PEG 1000 ATPS (K(D)=1.4) when it is completely located in the BMIM Cl upper phase of the ionic liquid ATPS (K(D)=180). The discrimination factor of the ionic liquid system and its intrinsic hydrophobicity were respectively found three times higher and ten times lower than the respective values of the PEG 1000 ATPS.     

Pressurized liquid extraction with in-cell clean-up followed by gas chromatography-tandem mass spectrometry for the selective determination of parabens and triclosan in indoor dust

This work studied the possibility of using polyethyleimine (PEI) as an affinity ligand for the purification of plasmid DNA (pDNA) from alkaline lysates using aqueous two-phase systems (ATPSs). The goal was to find conditions under which this cationic polymer could steer the partition of pDNA to the phase where less impurities accumulate. In poly(ethylene glycol) (PEG)/ammonium sulphate systems, neither free nor PEGylated PEI (pPEI) were able to change the partition of pDNA. This is probably due to the high salt concentration present in these systems that impair the interaction between pDNA and PEI. In PEG 3350/dextran 110 systems, the desired effect could be observed but 0.2-0.5M ammonium sulphate had to be added to prevent the co-partition of RNA to the same phase. These results were used to develop a methodology to obtain polyplexes from alkaline lysates in a two-step ATPSs extraction process. In the first step, a PEG 600/ammonium sulphate system is used to remove most impurities to the top phase. The pDNA-containing bottom phase is then isolated and contacted with a second PEG 3350/dextran 110 system supplemented with a small amount of pPEI (0.2%). Plasmid yield was 100% and the final preparation had no RNA and only small amounts of contaminant protein. Additionally, pDNA was obtained in the form of 53nm-sized polyplexes which are likely to suit specific gene delivery applications.     

Interfacial Tension of Polyethylene Glycol/Potassium Phosphate Aqueous Two-Phase Systems

Abstract

The interfacial tension of Polyethylene glycol (PEG)/potassium phosphate two-phase systems was measured by the rotating drop method. The interfacial tension was as low as 0.001 dyne/cm and increased with increases in the total concentrations of both PEG and potassium phosphate in two-phase systems. The increase in the interfacial tension was a function of the concentration differences of PEG and potassium phosphate between the top and the bottom phases which was confirmed by the tie line analysis. The interfacial tension was affected also by the molecular weigth of PEG. At low PEG molecular weights, the increase in the molecular weight greatly increased the interfacial tension, but at high molecular weights, the interfacial tension varied less with the molecular weight.     

"On the Collander equation": protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of randomly selected proteins were measured in aqueous two-phase systems (ATPSs) formed by different combinations of Dextran-75 (Dex), Ficoll-70, polyethylene glycol-8000 (PEG), hydroxypropyl starch-100 (PES), and Ucon50HB5100 (Ucon, a random copolymer of ethylene glycol and propylene glycol) at particular polymer concentrations, all containing 0.15M NaCl in 0.01 M phosphate buffer, pH 7.4. Most of the proteins in the PEG-Ucon system precipitated at the interface. In the other ATPSs, namely, PES-PEG, PES-Ucon, Ficoll-PEG, Ficoll-Ucon, and in Dex-PEG and Dex-Ucon described earlier the distribution coefficients for the proteins were correlated according to the solvent regression equation: lnKi=aiolnKo+bio, where Ki and Ko are the distribution coefficients for any protein in the ith and oth two-phase systems. Coefficients aio and bio are constants, the values of which depend upon the particular compositions of the two-phase systems under comparison.     

Study of phase behaviour for the aqueous two-phase polymer–polymer systems using the modified UNIQUAC-NRF model

A modified form of the UNIQUAC-NRF activity coefficient model was used to study the phase behaviour of the aqueous two-phase systems (ATPSs) of polyethylene glycol (PEG) and dextran (DEX) with different molecular weights at various temperatures. In the proposed model, a ternary interaction parameter was added to the expression for the excess Gibbs free energy and, in turn, to the corresponding activity coefficient rendered by the UNIQUAC-NRF model. The combinatorial part of the new model takes the same form as that of the original UNIQUAC model and the residual part considers the nonrandomness and also the binary and the ternary interactions among the molecules in mixtures of PEG, DEX, and water. The results show that the new model can more accurately correlate the experimental data for the systems studied in this work than those obtained from the original UNIQUAC and the UNIQUAC-NRF activity coefficient models. In order to favourably compare the results the same minimisation procedure and the same experimental data were used for the models studied in this work. The results for the Root Mean Square Deviations (RMSD) produced by the three UNIQUAC-based models are also reported.     

Atomic Absorption Determination of Some Amino Acids Containing a Sulphur Group

Abstract

A simple and rapid atomic absorption method for the determination of some amino acids containing a sulphur group is described. The studied compounds are cysteine, cystine and methionine. The method is based on the addition of an excess of mercury (II) chloride in phosphate buffer, pH 9, forming a white precipitate; the unreacted mercury is separated by centrifugation and the mercury ions in both precipitate and filtrate are determined by atomic absorption spectrophotometry at 253.6 nm. The concentration of the studied amino acids is then indirectly determined from a calibration curve for standard mercury (II) chloride solution.

The procedure has been successfully applied to the assay of the pharmaceutical preparations of the studied drugs after thin-layer chromatographic separation. The results of the studied compounds compare favourably with the official methods.     

Drop formation in aqueous two-phase systems

Extraction using aqueous two-phase systems (ATPSs) is a versatile technique for the downstream processing of various proteins/enzymes. The study of drop formation deals with the fundamental understanding of the behavior of liquid drops under the influence of various external body as well as surface forces. These studies provide a basis for designing of the extractions in column contactors in which liquid drops play a major role. Most of the drop formation studies reported so far is restricted to aqueous-organic systems. ATPSs, differ from aqueous-organic systems in their physical properties. In view of this, an attempt was made to develop a model for drop formation in ATPSs adopting the information available on aqueous-organic systems. In order to validate the model, experiments were performed by using polyethylene glycol (PEG)/salt systems of different phase compositions at various flow rates. At low flow rates the single stage model and at high flow rates the two stage model are able to predict the drop volume during its formation from tip of capillary. The experimental results were found to agree reasonably well with those predicted by the model.     

Swelling equilibrium of poly(acrylamide) gels in aqueous salt and polymer solutions

The influence of some single salts (NaCl, KCl, Na₂HPO₄ and K₂HPO₄) and poly(ethylene glycol) (PEG) on the swelling of aqueous poly(arcylamide)-gels was studied at 25°C in more than 600 experiments. The chlorides and phosphates cause a different behavior at high salt concentrations: The polyacrylamide gels swell in aqueous solutions of sodium and potassium chloride whereas they shrink when chloride ions are substituted by hydrogen phosphate ions. These differences are due to differences in the interactions of chloride and hydrogen phosphate ions with the network groups. In aqueous solutions of poly(ethylene glycol) the gels shrink continuously with increasing polymer concentration. At constant PEG mass fraction in the liquid phase, the swelling of the gel decreases with increasing molecular weight of PEG. The experimental results (degree of swelling, partitioning of solutes to the coexisting phases) are correlated by combining a model for the Gibbs excess energy for aqueous systems of polymers and electrolytes with a modification of the phantom-network theory. The correlation gives a good agreement with the experimental data for the degree of swelling, whereas in most cases, there is only a qualitative agreement for the partitioning of the solutes.     

Extraction of the antimicrobial peptide cerein 8A by aqueous two-phase systems and aqueous two-phase micellar systems

Cerein 8A is an antimicrobial peptide with potential application against food spoilage and pathogenic bacteria. The partitioning of cerein 8A was investigated in two liquid-liquid extraction systems that are considered promising for bioseparation and purification purposes. Aqueous two-phase systems (ATPSs) were prepared with polyethylene glycol (PEG) and inorganic salts, and the addition of NaCl was investigated in this system. The best results concerning partition coefficients (K (b)) were obtained with PEG?+?ammonium sulphate, and K (b) value significantly increases when NaCl was added. Cerein 8A was effectively extracted into the micelle-rich phase in a 4% Triton X-114 medium. Recovery yield was higher for ATPS compared to micellar systems. Cerein 8A can be isolated from a crude suspension containing the bioactive molecule by ATPSs. Successful implementation of peptide partitioning represents an important step towards developing a low-cost effective separation method for cerein 8A.     

Ion-pair LC of sugars at alkaline pH and pulsed electrochemical detection

Summary Sugars, sugar acids, amino sugars and oligomers are separated as ion-pairs with hydrophobic counter ions at alkaline pH using PLRP-S and Hypercarb as solid phases. Important parameters for regulation of retention and selectivity are nature and concentration of the counter ion, pH (hydroxide concentration) and temperature. Reversals in the elution order wer obtained in some cases. Oligosaccharides are highly retained in these systems. The addition of organic modifiers to the mobile phase for elution of the solutes were found to interfere with the pulsed electrochemical detection (PED). Anions added to the mobile phase compete with the solutes for ion-pair retention, hence, decreasing the capacity factors, and phosphate could be used for this purpose in the separation of maltooligomers (M2-M10) from corn syrup.     

Surface modification of proteins activation of monomethoxy-polyethylene glycols by phenylchloroformates and modification of ribonuclease and superoxide dismutase

A single-step method of activation of monomethoxypolyethylene glycols suitable for its binding to polypeptides and proteins is proposed. Based on the reaction with 2,4,5-trichlorophenylchloroformate or p-nitrophenylchloroformate, it gives reactive PEG-phenylcarbonate derivatives. The PEG intermediate is stable on storage, the activating group is easily quantified,and the reaction with amino acid and proteins proceeds rapidly at pH near neutrality. The PEG derivatization of enzymes with this procedure is less inactivating than those previously reported. Ribonuclease and superoxide dismutase were modified and the effect of (a) bound polymer on clearance time in rats, (b) antibody recognition, and (c) on the enzymatic activity toward low and high molecular weight substrates were studied.     

Analysis of acrylamido-buffers for isoelectric focusing by capillary zone electrophoresis

Immobilized pH gradients use a series of weak acrylamido acids and bases (Immobiline) to create a pH gradient along the separation axis. These buffers can be degraded in water by two mechanisms: (i) hydrolysis of the amido bond, with generation of free acrylic acid and either an amino acid or a diamine; (ii) autopolymerization to oligomers and/or n-mers. In order to check for these degradation products, different capillary zone electrophoresis systems for analysis of all Immobilines have been devised. The acidic compounds are resolved in 100 mM acetate, pH 4.0, whereas the alkaline Immobilines are separated in 50 mM phosphate buffer, pH 7.7 (or pH 7.2 for the weaker species). Polymers of alkaline Immobilines are resolved in 50 mM phosphate buffer, pH 2.5, in 1% Ficoll-400. All Immobilines are detected underivatized, by their adsorption at 214 or 254 nm. A calibration curve has been constructed for quantification of acrylic acid contamination. As little as 1 mol% of acrylic acid contamination in Immobiline solutions can be detected, with a sensitivity limit below 0.2 mM (at the injection port).     

铟在聚乙二醇-硫酸铵双水相体系中的分配

研究了稀散元素铟在有配合剂PAR（4－（2－吡啶偶氮）－间苯二酚）存在和无PAR存在的聚乙二醇PEG－（NH4）2SO4双水相体系中的分配行为。讨论了酸度、PEG分子量、PEG浓度及温度等因素对铟分配比的影响，发现酸度对分配比的影响最大，随着PEG分子量的增加及温度的上升分配比逐渐增大；随着PEG浓度的增加分配比逐渐减小。