Aqueous Two-phase Solvent Systems Research Articles

Green and sustainable extraction of phycocyanin from Spirulina platensis by temperature-sensitive polymer-based aqueous two-phase system and mechanism study

In this study, a sustainable and environmentally friendly method was developed for the enrichment and purification of phycocyanin from Spirulina platensis. This was achieved by utilizing a temperature-sensitive polymer, Pluronic F68, in an aqueous two-phase solvent system. The phase behavior of the temperature-sensitive polymer-based biphasic system was evaluated. The extraction conditions were optimized by both single-factor experiments and response surface methodology. Under the optimal conditions, the upper polymer-rich phase was recycled for sustainable phycocyanin extraction, resulting in a grade of 3.23 during the third extraction cycle. Pluronic F68 could be efficiently recovered and reused during the extraction process. The interaction mechanism between Pluronic F68 and phycocyanin was systematically studied using FT-IR and fluorescence analysis. This was further complemented by static and dynamic calculation of molecular motion through molecular docking and molecular dynamics simulation, indicating that hydrophobic segment of Pluronic F68 played a key role in the binding process with phycocyanin.

Countercurrent chromatography as a frontier tool to discover new mechanical roles in liquid-liquid phase separation of cellular biomolecules.

The development of countercurrent chromatography (CCC) technology enabled us to achieve higher peak resolutions and more shortened separation times even for protein separation using aqueous two-phase solvent systems composed of polyethylene glycol and inorganic salts (or dextrans). By eliminating the solid support matrix, all analytes can be recovered from the coiled column after the separation is completed. Recently, it has been found that droplets of biomolecules formed by liquid-liquid phase separation in cells closely relate to the transcription, regulation of signal transduction, and formation of amyloids. Meanwhile, although CCC is a separation technique based on liquid-liquid partitioning of analytes between two immiscible phases, the mechanism of separation could suggest some idea concerning the formation of biomolecule droplets in cells. This article describes the recent advances in the CCC apparatus, the coiled separation column, the choice of a suitable two-phase solvent system, and the application to separation and purification of bioactive macromolecules such as proteins and enzymes, and also discusses the possibility of CCC as a tool to reveal new mechanical roles of biomolecule droplets in the cellular environments.

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.

Spiral Countercurrent Chromatography Enrichment, Characterization, and Assays of Carbon Nanotube Chiralities for Use in Biosensors.

Single-walled carbon nanotubes (SWCNTs) are synthetic materials that hold great promise for electronics that are smaller and more versatile than the current silica-based technologies. But as-produced SWCNTs are generally a mixture of nanotubes with different structures that have vastly different properties. Separating these SWCNTs from multiwalled and metallic carbon nanotubes is vital to explore their individual properties and commercial utility ranging from optics to semiconductors. Compounding the problem of commercial investigation is that the semiconducting SWCNTs are also a mixture of different diameters and/or chiralities with different properties. Analyzing properties of enriched semiconducting SWCNT chiralities has only recently been possible through separation techniques such as aqueous two-phase solvent systems. Our study illustrates a semipreparative spiral countercurrent chromatography (CCC) separation of a commercial mixture of SWCNTs into distinct enriched fractions. A new mixer–settler spiral disk rotor was applied in this study, in which we compare the enriched SWCNTs for their effectiveness in biosensors with a high-throughput model assay, followed by antibody-mediated detection of Escherichia coli. Our results demonstrate that CCC-enriched responsive SWCNTs for biosensors can be used in our model assay, as well as for the detection of E. coli. To date, we believe that this is the first study along with Liu et al. [Chirality-controlled synthesis of single-wall carbon nanotubes using vapour-phase epitaxy. Nat. Commun.2012, 3, 119923149724] to demonstrate a specific utility of separated SWCNT species.

Design of a coil satellite centrifuge and its performance on counter-current chromatographic separation of 4-methylumbelliferyl sugar derivatives with polar organic–aqueous two-phase solvent systems

A new high-speed counter-current chromatograph, named coil satellite centrifuge (CSC), was designed and fabricated in our laboratory. The CSC apparatus produces the satellite motion such that the coiled column simultaneously rotates around the sun axis (the angular velocity, ω1), the planet axis (ω2) and the satellite axis (the central axis of the column) (ω3). In order to achieve this triplicate rotary motion without twisting of the flow tube, the rotation of each axis was determined by the following formula: ω1=ω2+ω3. This relation enabled to lay out the flow tube without twisting by the simultaneous rotation of three axes. The flow tube was introduced from the bottom side of the apparatus into the sun axis of the first rotary frame reaching the upper side of the planet axis and connected to the column in the satellite axis. The performance of the apparatus was examined on separation of 4-methylumbelliferyl (MU) sugar derivatives as test samples with organic–aqueous two-phase solvent systems composed of ethyl acetate/1-butanol/water (3:2:5, v/v) for lower phase mobile and (1:4:5, v/v) for upper phase mobile. With lower phase mobile, five 4-MU sugar derivatives including β-d-cellobioside (Cel), β-d-glucopyranoside, α-d-mannopyranoside, β-d-fucopyranoside and α-l-fucopyranoside (α-l-Fuc) were separated with the combined rotation around each axis at counterclockwise (CCW) (ω1) – CCW (ω2) – CCW (ω3) by the flow tube distribution. With upper phase mobile, three 4-MU sugar derivatives including α-l-Fuc, β-d-galactopyranoside and Cel were separated with the combined rotation around each axis at clockwise (CW) (ω1) – CW (ω2) – CW (ω3) by the flow tube distribution. A series of experiments on peak resolution and stationary phase retention revealed that better partition efficiencies were obtained at the flow rate of 0.5mL/min (column 1) and 0.8mL/min (column 2) for lower phase mobile and 0.2mL/min (column 1) and 0.4mL/min (column 2) for upper phase mobile when using the left-handed multilayer coil (total capacity: 57.0mL for column 1 and 75.0mL for column 2) under the rotation speeds of approximately ω1=300rpm, ω2=150rpm and ω3=150rpm.

Hydrophilic organic/salt-containing aqueous two-phase solvent system for counter-current chromatography: A novel technique for separation of polar compounds

Hydrophilic organic/salt-containing aqueous two-phase system composing of ethanol, water and ammonium sulfate for separation polar compounds was investigated on multilayer coil associated with J-type HSCCC devices. Compared to the classical polar solvent system based on 1-butanol–water or PEG1000–ammonium sulfate–water, the water content of upper phase in ethanol–ammonium sulfate–water systems was from 53.7% to 32.8% (wt%), closed to PEG1000–ammonium sulfate–water aqueous two-phase systems and higher than 1-butanol–water (22.0%, wt%). Therefore, the polarity of ethanol–ammonium sulfate–water is in the middle of 1-butanol–water and PEG–ammonium sulfate–water system, which is quite good for separating polar compounds like phenols, nucleosides and amino acids with low partition coefficient in 1-octanol–water system. The retention of stationary phase in four elution mode on type-J counter-current chromatography devices with multilayer coil column changed from 26% to 71%. Hydrodynamic trend possess both intermediate and hydrophilic solvent system property, which closely related to the composition of solvent system. The applicability of this system was demonstrated by successful separation of adenosine, uridine guanosine and cytidine.

Preparative separation of two subsidiary colors of FD&C Yellow No. 5 (Tartrazine) using spiral high-speed counter-current chromatography

Specifications in the U.S. Code of Federal Regulations for the color additive FD&C Yellow No. 5 (Color Index No. 19140) limit the level of the tetrasodium salt of 4-[(4′,5-disulfo[1,1′-biphenyl]-2-yl)hydrazono]-4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylic acid and that of the trisodium salt of 4,4′-[4,5-dihydro-5-oxo-4-[(4-sulfophenyl)hydrazono]-1H-pyrazol-1,3-diyl]bis[benzenesulfonic acid], which are subsidiary colors abbreviated as Pk5 and Pk7, respectively. Small amounts of Pk5 and Pk7 are needed by the U.S. Food and Drug Administration for confirmatory analyses and for development of analytical methods. The present study describes the use of spiral high-speed counter-current chromatography (HSCCC) to separate the closely related minor components Pk5 and Pk7 from a sample of FD&C Yellow No. 5 containing ∼3.5% Pk5 and ∼0.7% Pk7. The separations were performed with highly polar organic/high-ionic strength aqueous two-phase solvent systems that were chosen by applying the recently introduced method known as graphic optimization of partition coefficients (Zeng et al., 2013). Multiple ∼1.0g portions of FD&C Yellow No. 5 (totaling 6.4g dye) were separated, using the upper phase of the solvent system 1-butanol/abs. ethanol/saturated ammonium sulfate/water, 1.7:0.3:1:1, v/v/v/v, as the mobile phase. After removing the ammonium sulfate from the HSCCC-collected fractions, these separations resulted in an enriched dye mixture (∼160mg) of which Pk5 represented ∼46% and Pk7, ∼21%. Separation of the enriched mixture, this time using the lower phase of that solvent system as the mobile phase, resulted in ∼61mg of Pk5 collected in fractions whose purity ranged from 88.0% to 92.7%. Pk7 (20.7mg, ∼83% purity) was recovered from the upper phase of the column contents. Application of this procedure also resulted in purifying the major component of FD&C Yellow No. 5 to >99% purity. The separated compounds were characterized by high-resolution mass spectrometry and several 1H and 13C nuclear magnetic resonance spectroscopic techniques.

Partition efficiencies of newly fabricated universal high-speed counter-current chromatograph for separation of two different types of sugar derivatives with organic–aqueous two-phase solvent systems

A new design of universal high-speed counter-current chromatograph (HSCCC) was fabricated in our laboratory. It holds a set of four column holders symmetrically around the rotary frame at a distance of 11.2cm from the central axis. By engaging the stationary gear on the central axis of the centrifuge to the planetary gears on the column holder shaft through a set of idle gears, two pairs of diagonally located column holders simultaneously rotate about their own axes in the opposite directions: one forward (type-J planetary motion) and the other backward (type-I planetary motion) each synchronously with the revolution. Using the eccentric coil assembly, partition efficiencies produced by these two planetary motions were compared on the separation of two different types of sugar derivatives (4-methylumbelliferyl and 5-bromo-4-chloro-3-indoxyl sugar derivatives) using organic–aqueous two-phase solvent systems composed of n-hexane/ethyl acetate/1-butanol/methanol/water and aqueous 0.1M sodium tetraborate, respectively. With lower phase mobile, better peak resolution was obtained by the type-J forward rotation for both samples probably due to higher retention of the stationary phase. With upper phase mobile, however, similar peak resolutions were obtained between these two planetary motions for both sugar derivatives. The overall results indicate that the present universal HSCCC is useful for counter-current chromatographic separation since each planetary motion has its specific applications: e.g., vortex CCC by the type-I planetary motion and HSCCC by the type-J planetary motion both for separation of various natural and synthetic products.

Purification of a fucoidan from kelp polysaccharide and its inhibitory kinetics for tyrosinase

High-speed countercurrent chromatography (HSCCC) was used to separate kelp polysaccharide. HSCCC was performed using an aqueous two-phase solvent system composed of PEG1000–K2HPO4–KH2PO4–H2O (0.5:1.25:1.25:7.0, w/w) by eluting a lower aqueous phase at 2.0mL/min at 600rpm, yielding two separate fractions, KPS-1 and KPS-2. The KPS-2 fraction was further purified by DEAE-Sepharose fast flow anion-exchange column chromatography to provide 3 fractions, KPS-2-1, KPS-2-2 and KPS-2-3. GPC–HPLC analysis indicated that KPS-2-1 fraction was a purified fucoidan. FT-IR analysis showed that KPS-2-1 was a sulphated polysaccharide. An analysis of enzymatic kinetics showed that the purified fucoidan was a competitive inhibitor of tyrosinase toward l-tyrosine, and the inhibitory constant Ki obtained from double-reciprocal plots was 0.9907mg/mL.

Organic high ionic strength aqueous two-phase solvent system series for separation of ultra-polar compounds by spiral high-speed counter-current chromatography.

Existing two-phase solvent systems for high-speed countercurrent chromatography cover the separation of hydrophobic to moderately polar compounds, but often fail to provide suitable partition coefficient values for highly polar compounds, such as sulfonic acids, catecholamines and zwitter ions. The present paper introduces a new solvent series which can be applied for the separation of these polar compounds. It is composed of 1-butanol, ethanol, saturated ammonium sulfate and water at various volume ratios and consists of a series of 10 steps which are arranged according to the polarity of the solvent system so that the two-phase solvent system with suitable K values for the target compound(s) can be found in a few steps. Each solvent system gives proper volume ratio and high density difference between the two phases to provide a satisfactory level of retention of the stationary phase in the spiral column assembly. The method is validated by partition coefficient measurement of four typical polar compounds including methyl green (basic dye), tartrazine (sulfonic acid), tyrosine (zwitter ion) and epinephrine (a catecholamine), all of which show low partition coefficient values in the polar 1-butanol-water system. The capability of the method is demonstrated by separation of three catecholamines.

Application of centrifugal precipitation chromatography and high‐speed counter‐current chromatography equipped with a spiral tubing support rotor for the isolation and partial characterization of carotenoid cleavage‐like enzymes in Enteromorpha compressa (L.) Nees

Centrifugal precipitation chromatography and a high-speed counter-current chromatography system equipped with a spiral tubing support rotor (spHSCCC) were successfully applied for the identification and isolation of carotenoid cleavage-like enzymes from Enteromorpha compressa (L.) Nees. This is the first study separating active enzymes from a complex natural matrix by spHSCCC. The target enzymes were identified after fractionation of the proteins in an acetone Tris-buffer gradient by centrifugal precipitation chromatography. Also, an aqueous two-phase solvent system consisting of PEG 1000 and mono- and dibasic potassium phosphate was used for the isolation of the enzymes by spHSCCC. The purified fractions contained two proteins of 65 and 72 kDa, respectively. The enzymes could cleave β-carotene and β-apo-8'-carotenal to produce β-ionone.

Effect of Elution Modes on Protein Separation by Cross-Axis Coil Planet Centrifuge with Two Different Types of Coiled Columns

ABSTRACT Effect of elution modes on protein separation was investigated using cross-axis coil planet centrifuge (cross-axis CPC) with two different types of coiled columns, i.e., eccentric coil and toroidal coil assemblies. Myoglobin and lysozyme were separated with an aqueous two-phase solvent system composed of 12·5% (w/w) polyethylene glycol 1000 and 12·5% (w/w) dibasic potassium phosphate. The substantial effect of elution modes was observed by the toroidal coil, while the negative result was given at the eccentric coil. Using the toroidal coil, higher peak resolution of proteins was attained at the tail to head elution mode. In the outward lower phase mobile elution mode, the satisfactory separation was obtained by both eccentric coil and toroidal coil assemblies. However, in the inward upper phase mobile elution mode, the toroidal coil produced a broad and asymmetric myoglobin peak. The analysis using polyacrylamide gel electrophoresis revealed that the toroidal coil partially separated the components originally present in the myoglobin sample. As the result, a modified equation was devised to express the peak resolution (Rs) using the lysozyme peak. The overall results indicated that the toroidal coil produced better partition efficiency than the eccentric coil under the optimized experimental condition including the direction of the Coriolis force acting on the mobile phase in the toroidal coil.

Purification of Proteins with Aqueous Two-Phase Solvent Systems by Countercurrent Chromatography

Aqueous polymer phase systems, introduced by Albertsson1 in the 1950s, have been used for partitioning a variety of macromolecules and cell particles which include proteins,2-6 nucleic acid,7-10 cell organelles,11 viruse,12,13 bacterial cells,14,15 etc. Using these solvent systems, several different approaches have been made for performing purification of biological samples, such as single step partitioning,1 repetitive batch extraction, countercurrent distribution (CCD)16,17 and countercurrent chromatography (CCC).18-21 Among these techniques, CCC is considered to be most effective in terms of partition efficiency and separation times.

Enantiomeric Separation of Commercial D,L-Kynurenine with an Aqueous Two-Phase Solvent System by Cross-Axis Coil Planet Centrifuge

Commercial D,L-kynurenine was resolved by high-speed countercurrent chromatography using a cross-axis coil planet centrifuge. The separation was performed with an aqueous-aqueous polymer phase system composed of 10% (w/w) polyethylene glycol 8000 and 5% (w/w) dibasic sodium phosphate containing 6% (w/w) bovine serum albumin as a chiral selector. The lower phosphate-rich mobile phase eluted the L-enantiomer first followed by the D-enantiomer. The peak resolution was 0.94 for 2.5 mg of the sample. The separation was completed within 3.5 h.

Water-soluble organometallic compounds. 5. The regio-selective catalytic hydrogenation of unsaturated aldehydes to saturated aldehydes in an aqueous two-phase solvent system using 1,3,5-triaza-7-phosphaadamantane complexes of rhodium

The water-soluble phosphine complex of Rh(I), [Rh(PTAH)(PTA) 2Cl]Cl( 1), has been synthesized from RhCl 3 and 1,3,5,-triaza-7-phosphaadamantane (PTA) in 96% ethanol. This complex is an effective catalyst for the regioselective reduction of unsaturated aldehydes to saturated aldehydes. The rate of hydrogenation of trans-cinnamaldehyde with sodium formate as reductant was studied as a function of catalyst, substrate, and sodium formate concentration. The presence of an excess of PTA was found to inhibit hydrogenations completely. This reaction was also found to be partially inhibited by cyclo-octatetraene and Hg(0), leading to the conclusion that both heterogenous and homogenous mechanisms are operating. Recycling experiments show complex 1 to be quite robust, with minimal leaching into the organic phase in a biphasic system. The complex resulting from the addition of an excess of PTA to 1, [Rh(PTAH) 3(PTA)Cl]Cl 3 ( 2) has been prepared and structurally characterized by a single crystal X-ray diffraction study. 31P and 1H NMR, IR and UV-VIS spectroscopy and pH titrametric measurements were employed to study the reactivity of the various rhodium PTA complexes in aqueous solutions.

Water-soluble organometallic compounds. 4. Catalytic hydrogenation of aldehydes in an aqueous two-phase solvent system using a 1,3,5-triaza-7-phosphaadamantane complex of ruthenium

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTWater-soluble organometallic compounds. 4. Catalytic hydrogenation of aldehydes in an aqueous two-phase solvent system using a 1,3,5-triaza-7-phosphaadamantane complex of rutheniumDonald J. Darensbourg, Ferenc Joo, Michael Kannisto, Agnes Katho, Joseph H. Reibenspies, and Donald J. DaigleCite this: Inorg. Chem. 1994, 33, 2, 200–208Publication Date (Print):January 1, 1994Publication History Published online1 May 2002Published inissue 1 January 1994https://doi.org/10.1021/ic00080a006RIGHTS & PERMISSIONSArticle Views838Altmetric-Citations129LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (966 KB) Get e-AlertsSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Purification of Uridine Phosphorylase from Crude Extracts of Escherichia Coli Employing High-Speed Countercurrent Chromatography With an Aqueous Two-Phase Solvent System

Abstract This is a preliminary report on a novel liquid-liquid parti tioning chromatography method for the purification of recombinant protein, uridine phosphorylase (UrdPase). This system utilizes an aqueous two-phase solvent system, consisting of polyethylene glycol and phosphate buffer, with a newly developed high-speed countercurrent centrifuge (X-axis, XLL type). The system is capable of purifying crude recombinant UrdPase in an efficient manner.

Comparison of Several Aqueous two Phase Solvent Systems (ATPS) for the Fractionation of Biopolymers by Centrifugal Partition Chromatography (CPC)

Abstract Aqueous Two Phase Solvent Systems {ATPS} have been tested in a Centrifugal Partition Chromatograph (Sanki, Kyoto, Japan), to check for stability, backpressure, noiseless baseline and efficiency. From our experiments we conclude that ATPS are compatible with CPC, in particular the stationary phase is very stable and does not require a high rotational speed to stabilize it (1300 rpm was commonly used); but, the efficiency of the CPC apparatus decreases markedly, due to the high viscosity of ATPS as compared to classical biphasic solvent systems. Based en our results, new partition cartridges have been designed, which minimize the bulk volumes and considerably increase the interface, or favor the mixing step of the two phases by an ad-hoc design of both the inlet ducts and channels.