New phase supports for partition chromatography of biopolymers

Liquid-liquid partition chromatography of bio-polymers requires aqueous two-phase systems for reasons of sample solubility and stability. Such aqueous two-phase systems form when thermodynamically incompatible polymers are co-dissolved in water. The most common polymer combination providing a two-phase system at reasonably low polymer concentrations is the combination of poly(ethylene glycol) (“PEG”) and dextran (“DX”), detected and introduced for the separation of biopolymers and cells by Albertsson about 30 years ago. The application of this powerful system for liquid-liquid partition chromatography requires support materials with surfaces able to immobilize selectively one of the two aqueous phases. This phase immobilisation may be achieved by exploiting incompatibilities between the polymers dominating in the phases and the hydrated support surface. Examples involving diol-modified silica and polyacrylamide coated diol-silica as support materials in aqueous PEG-DX and PEG-salt systems are presented. The application of such systems for the separation of biopolymers is demonstrated.     

Extension of the Wilson-NRF Gibbs Energy Model in Correlating Vapor-Liquid and Liquid-Liquid Phase Behavior of Polymer-Polymer Aqueous Two-Phase Systems

In this work, the proposed model by Pazuki et al. based on the Local Composition Concept (LCC), has been used in correlating the vapor-liquid phase behavior of polymer solutions and the liquid-liquid phase behavior of aqueous two-phase systems. The Flory-Huggins model has been used as the combinatorial part of the proposed model, as well as the model proposed by Pazuki et al. was considered as the residual term. The proposed model has been used in correlating the vapor-liquid phase behavior for a number of PEG-Water systems at constant temperature. The results obtained from the proposed model have been compared with those obtained from the Poly-NRTL and the Poly-Wilson models. The results showed that the proposed model can accurately correlate the VLE data for PEG-Water systems. Also, the proposed model has been used to obtain phase behavior of aqueous two-phase systems for PEG-DEX-Water systems. The results obtained from the proposed model have been compared with those obtained from the UNIQUAC and the UNIQUAC-NRF models. The results showed that the proposed model can accurately correlate liquid-liquid phase behavior of aqueous two-phase systems than the UNIQUAC and the UNIQUAC-NRF models.     

Countercurrent Chromatography Using a Toroidal Coil Planet-Centrifuge: A Comparative Study of the Separation of Organelles Using Aqueous Two-Phase Partition

Abstract

A toroidal coil planet centrifuge is described and compared with other countercurrent chromatography (CCC) and countercurrent distribution (CCD) techniques. The basis of separation is partition in aqueous two-phase polymer systems, with each method assessed by fractionating rat liver organelles. The size and ease of operation of the toroidal coil planet centrifuge gave significant advantages over conventional CCD systems achieving equivalent resolution in a fraction of the time.     

生物分子液-液相分离的物理化学机制

近年来,有关生物分子通过液-液相分离机制进行组织定位、功能调控的研究发展迅速。相分离产生的聚集体在众多细胞活动事件中发挥了关键作用。这些聚集体的生物功能是以相分离的物理化学性质为基础的。本文将从相分离聚集体的基本性质、相图、微观结构,相分离的统计热力学、实验和分子模拟研究等方面阐释相分离物理化学机制研究相关进展。对于生物分子相分离的重要功能体系进行了列举和归纳,收集了相分离研究的模式体系,探讨了生物分子相分离的生物功能同物理化学机制之间的关系,总结了生物分子相分离的调控机制和调控分子的设计方法,并对生物分子相分离物理化学机制研究的未来发展方向进行了展望。     

Partition of macromolecules and cell particles in aqueous two-phase systems based on hydroxypropyl starch and poly(ethylene glycol)

The partition behavior of proteins, nucleic acids, cell membranes, cell organelles and whole cells has been studied in liquid-liquid two-phase systems composed of water, poly-(ethylene glycol), and an hydroxypropyl starch. The properties of the systems are in many respects comparable with the traditional poly(ethylene glycol)-dextran systems, but the cost is reduced to around one-fifth.     

Assay of N-Propylajmaline in Blood Plasma by Ion-Pair Liquid-Liquid Chromatography

Abstract

A sensitive method for assay of N-propylajmaline (prajmaline) in human plasma is described. The quaternary ammonium compound exists as a pair of stereoisomers, which are isolated and separated by ion-pair liquid-liquid chromatography on microporous silica particles. An aqueous solution containing perchloric acid and sodium perchlorate is used as stationary phase and a mixture of butanol, dichloroethane and hexane as mobile phase. The procedure involves ion-pair extraction from plasma and evaporation prior to the chromatographic separation. Selective detection is achieved by using a fluorescence detector. The method allows assay of concentrations down to 10 pmol of the two forms of prajmaline in 1 ml of plasma with a relative standard deviation below 5 %.     

Mutual Separation of Lanthanoid Elements by Centrifugal Partition Chromatography

Abstract

Multistage separation based on liquid-liquid extraction has been investigated by means of centrifugal partition chromatography (CPC). A kerosene solution of 2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester (EHPA) was employed as a stationary phase without any solid support. Metal ions eluted by the aqueous mobile phase were detected by the post-column reaction with Arsenazo III. The retention volumes are approximately linear with the distribution ratios of metals. The mutual separation of adjacent lanthanoids was accomplished by CPC.     

Aqueous two-phase systems in biochemical recovery

The design and application of aqueous two-phase systems for recovery of proteins is reviewed. Reference is made to the properties of polymers and salt forming the systems and their influence on phase separation and partition. The properties of systems important for the design of purification strategies are discussed in relation to the surface properties of proteins. Strategies for the modification of systems for bioaffinity partition are considered including the choice of carrier in terms of molecular type and properties, or by particulate addition. Finally, the scaleup of partition is considered, and the reasons for currently limited adoption at larger scales are elucidated.     

Partition countercurrent chromatography in inorganic analysis

Summary Partition countercurrent chromatography can be used for the concentration and separation of elements of similar properties in aqueous solution — extradant systems. The angle of wetting of the column wall with the components of a two-phase liquid system has a considerable effect on the retention of the stationary phase. The method was used for the separation of orthophosphate and pyrophosphate (extradant — solution of dinonyltin dichloride in methylisobutylketone), cesium and strontium (solution of cobalt dicarbollide in nitrobenzene) and rare earth elements (di-2-ethyhexylphosphoric acid in decane).

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Gegenstrom-Verteilungs-Chromatographie in der anorganischen Analyse

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Dedicated to Prof. Dr. G. Tölg on the occasion of his 60th birthday     

Effective extraction of tylosin and spiramycin from fermentation broth using thermo-responsive ethylene oxide/propylene oxide aqueous two-phase systems

Recyclable aqueous two-phase systems with thermo-responsive phase-forming materials have been employed to separate macromolecules; however, these systems have achieved very limited separation efficiency for small molecules, such as antibiotics. In this study, aqueous two-phase systems composed of the ethylene oxide/propylene oxide copolymer and water were developed to extract alkaline antibiotics from the fermentation broth. In the aqueous two-phase systems with an ethylene oxide ratio of 20 and propylene oxide ratio of 80, the partition coefficients of tylosin and spiramycin reached 16.87 and 20.39, respectively, while the extraction recoveries were 70.67 and 86.70%, respectively. Coupled with mechanism analysis, we demonstrated the feasibility of extracting alkaline antibiotics using this aqueous two-phase system, especially for 16-membered macrolide antibiotics. The molecular dynamic simulation was employed to visualize the process of dual-phase formation and the partition behavior of antibiotics in an aqueous two-phase system. The dynamic simulation revealed the binding energy between the antibiotic and ethylene oxide/propylene oxide copolymers, which provides a simple indicator for screening suitable antibiotics in aqueous two-phase systems. Our recyclable aqueous two-phase systems provide a robust approach for the extraction of 16-membered macrolide antibiotics with ease of operation and high recovery rates, which is appropriate for large-scale extraction in the fermentation industry.     

Partition behavior and partial purification of hexokinase in aqueous two-phase polyethylene glycol/citrate systems

This study dealt with the partition behavior and partial purification of hexokinase (HK) from baker’s yeast by liquid-liquid extraction using aqueous two-phase polyethylene glycol (PEG)/citrate systems. First, we investigated the effect of agitation type (vortex and 8 rpm rotation) on the stability of the system, and then the effects of sodium citrate concentration, PEG concentration, and molar mass of PEG on the partition coefficient of this enzyme by using a 2⁵ factorial experimental design. The results of this factorial experiment showed the possibility of a partial purification of HK by using two extraction steps, since the enzyme preferentially migrated to the top phase and the total proteins (mainly contaminants) remained in the bottom phase. The purification factor (Pur _TOP) of the enzyme in the top phase was 1.87, and the partition coefficient of the total proteins (K _Prot ) was 0.47.     

Solvent generated liquid-liquid systems on unmodified silica

Summary The liquid-liquid distribution properties of three liquid-liquid systems, solvent generated on silicagel, were investigated by correlating retention volumes of solutes with their statically determined partition coefficients. The application of these phase systems for the prediction of partition coefficients from chromatographic data and the performance of solvent generated liquid-liquid columns were studied. The results show that liquid-liquid distribution is by far the dominating retention process for the investigated binary liquid-liquid systems. This allows the determination of liquid-liquid partition coefficients with an accuracy of about 5% from chromatographic data. Further the solvent generated liquid-liquid systems show a surprisingly good column performance with theoretical plate heights of 2 times the particle diameter at the minimum of the H/〈v〉 curve. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Effect of Coriolis force on counter-current chromatographic separation by centrifugal partition chromatography

The effect of Coriolis force on the counter-current chromatographic separation was studied using centrifugal partition chromatography (CPC) with four different two-phase solvent systems including n-hexane-acetonitrile (ACN); tert-butyl methyl ether (MtBE)-aqueous 0.1% trifluoroacetic acid (TFA) (1:1); MtBE-ACN-aqueous 0.1% TFA (2:2:3); and 12.5% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) dibasic potassium phosphate. Each separation was performed by eluting either the upper phase in the ascending mode or the lower phase in the descending mode, each in clockwise (CW) and counterclockwise column rotation. Better partition efficiencies were attained by the CW rotation in both mobile phases in all the two-phase solvent systems examined. The mathematical analysis also revealed the Coriolis force works favorably under the CW column rotation for both mobile phases. The overall results demonstrated that the Coriolis force produces substantial effects on CPC separation in both organic-aqueous and aqueous-aqueous two-phase systems.     

Electroextraction

Since both phases of aqueous two-phase systems are electrically conductive, application of electric fields in these systems gives rise to electrokinetic mass transfer of charged species. We have shown that an aqueous two-phase system can be used as a medium for electrophoretic separation with the liquid-liquid interface providing stability against convection and facilitating product recovery. Proteins have been directed into either the top or bottom phases of polyethylene glycol/dextran aqueous two-phase systems using 20–50 V/cm electric fields perpendicular to the phase interfaces. Binary protein mixtures have been separated in both batch and continuous modes by operating between isoelectric points and directing oppositely charged proteins into separate phases. Preliminary results on focusingE. coli cells into one phase while directing a proteinaceous product into the other phase indicate that two-phase electrophoresis may prove useful for commercial-scale recovery of proteins from fermentation broth. Recent studies in our laboratory indicate that the electrostatic potential profile near the interface of conducting liquid-liquid systems can influence electrophoretic migration of charged species across the interface. We have observed that phase systems with high Donnan potentials hinder electrophoretic mass transfer across liquid-liquid interfaces, whereas phase systems with low Donnan potentials do not.     

利用双水相分离回收离子液体的研究进展

研究离子液体的分离与回收对于减少离子液体对环境的影响、提高离子液体的利用效率、降低离子液体的应用成本、促进离子液体的工业应用具有重要的意义.本文重点综述了利用无机盐-离子液体双水相、糖-离子液体双水相、聚合物-离子液体双水相和CO2诱导的离子液体双水相技术分离回收离子液体的研究进展,分析了影响离子液体分离回收的关键因素,评价了不同离子液体双水相体系的优缺点,展望了该领域的发展方向及面临的挑战.     

Solvent-generated liquid-liquid chromatography with aqueous ternary systems

Summary It is shown that solvent-generated liquid-liquid chromatography can also be realized with aqueous ternary systems. One phase of such a liquid-liquid system is used as eluent and the column is packed with a porous solid support whose surface is better wetted by the other phase which is then generated dynamically on it by the mobile phase. The partition mode of retention was verified by linear regression of the chromatographic retention data and the partition coefficients measured at equilibrium. A further confirmation was obtained by the sharp change observed in the retention characteristics when approaching a composition of the mobile phase corresponding to the liquid-liquid equilibrium. The stationary phase is generated rather quickly needing between 10 and 100 times the column volume of mobile phase for equilibration depending on the magnitude of the change of composition of the mobile phase and the specific surface area of the solid support. The stability of solvent-generated LLC columns is very high allowing long term reproducibility of the selectivity coefficient of about 1 %. It is demonstrated that solvent-generated LLC offers a powerful means to optimize the separation of a mixture by chromatography. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1–5, 1984; including results already presented at the 5th International Symposium on Column Liquid Chromatography, Avignon, May 11–15, 1981.     

聚丙烯酰胺-聚乙二醇-水体系相图及丙烯酰胺单体在两相中的分配

聚丙烯酰胺(PAAm)和聚乙二醇(PEG)两种水溶液混合时能形成双水相体系,其中上层为PEG富集相,下层为PAAm和PEG的混合相.用凝胶渗透色谱(GPC)法和浊度滴定法研究了PAAm-PEG-H₂O双水相体系的相图,结果表明,随着PEG分子量的升高,体系的分相浓度下降.在PAAm-PEG20000-H₂O体系中,随着体系温度升高,分相浓度先下降后升高,55℃时分相浓度最低.丙烯酰胺(AAm)单体能在两相中发生相分配,分配系数随着PAAm浓度和平衡温度的增加而增大,随着PEG浓度的增加而下降.     

Separation of Biopolymers by Partition in Aqueous Two-Phase Systems

Abstract

Aqueous two-phase systems are useful tools for fractionation and studies of cell components including proteins and nucleic acids. The systems consist of two non-miscible liquid layers which both have high content of water. The purification achieved by partition of biosubstances between the two liquid phases depends on the possibility to steer the partition in a selective way. The partition can be affected by hydrophobic and affinity ligands, as well as charged groups/ bound to soluble polymers.     

Separations of flavonoids and alkaloids in medicinal herbs by high-speed counter-current chromatography

Counter-current chromatography is a new liquid-liquid partition chromatography without using solid support. Recently, the technique has been remarkably improved in both partition efficiency and separation time. In this paper the capability of this high-speed counter-current chromatography was demonstrated on separation of two sets of samples obtained from medicinal herbs: a synthetic mixture of 3'-hydroxygenkwanin, luteolin and apigenin was separated on a two-phase solvent system composed of chloroform-methanol-water (4:3:2, v/v/v) and a crude ethanol extract from Anisodus tangulicus (Maxin) Pasch on chloroform-0.07 M sodium phosphate (pH 6.4) (1:1, v/v). In the light of chromatograms obtained from these samples, advantages of high-speed counter-current chromatography over other chromatographic methods were discussed in terms of partition efficiency, peak resolution, separation time, sample loading capacity, etc.     

Improved spiral tube assembly for high-speed counter-current chromatography

The original spiral tube support (STS) assembly is improved by changing the shape of the tubing, with 1-cm presses perpendicularly along the length. This modification interrupts the laminar flow of the mobile phase. The tubing in the four return grooves to the center of the rotor is flattened by a specially made pressing tool to increase the number of spiral layers and decrease the dead space volume, thus increasing the column efficiency. The performance of this spiral tube assembly was tested in separations of dipeptides and proteins with suitable polar two-phase solvent systems. The results revealed that the present system yields high partition efficiency with a satisfactory level of stationary phase retention in a short elution time. The present high-speed counter-current chromatographic (HSCCC) system will be efficiently applied to a broad spectrum of two-phase solvent systems including aqueous–aqueous polymer phase systems (TPAS) which are used for separation of biopolymers such as proteins and nucleic acids.