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 mM 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.     

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.     

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.     

Counter-current chromatography of lipoproteins with a polymer phase system using the cross-axis synchronous coil planet centrifuge.

Lipoproteins were separated by counter-current chromatography using the type-XLL coil planet centrifuge. The separation was performed with a polymer phase system composed of 16% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 0.5 ml/min. About 5 ml of the sample solution containing approximately 150 mg of a lipoprotein mixture were loaded. High- and low-density lipoproteins were resolved within 12 h. Each component was detected by gel electrophoresis with oil red staining.     

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.     

Development and evaluation of a spiral tube column for counter-current chromatography

An improved type-J counter-current chromatography (CCC) planet centrifuge with two spiral tube columns (volume 2×15 mL, β value 0.3-0.7, tubing 0.8 mm id) was developed and evaluated for its retention ability of four typical different solvent systems including heptane-methanol (1:1, v/v) (A), hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) (B), n-butanol-acetic acid-water (4:1:5, v/v) (C), PEG1000-K(2)HPO(4)-water (12.5:12.5:75, w/w) (D) under eight different operation modes. The results indicated that the spiral tube column could significantly increase the retention of four typical solvent systems compared with a traditional multilayer coil column with similar parameters (volume 35 mL, β value 0.3-0.7, tubing 0.8 mm id). The retention of stationary phase (S(f)) for the less polar system (A) and moderately polar solvent system (B) can be increased by about 10%, and for the polar system (C) and aqueous two-phase system (ATPS) (D) by 30-40%. The preliminary applications of this spiral tube column to the separation of small molecular compounds such as moderately polar theaflavins, polar anthocyanins and dipeptides were successful. Acceptable resolution can be obtained between cytochrome c and myoglobin, lysozyme and myoglobin when it was applied on protein separation; however, it still needs to be improved with regard to its column efficiency.     

Mixer-settler counter-current chromatography with multiple spiral disk assembly

A novel system for performing high-speed counter-current chromatography has been developed for separation of biopolymers using polymer phase systems. The spiral disk assembly consisting of eight units, each equipped with over 300 mixer-settler sets, was constructed and performance evaluated in terms of retention of the stationary phase and separation efficiency. A series of experiments was performed with a polymer phase system composed of polyethylene glycol 1000 (12.5%, w/w) and dibasic potassium phosphate (12.5%, w/w) using two stable protein samples of myoglobin and lysozyme at various experimental conditions of flow rates and revolution speeds. The best results were obtained with revolution speeds of 800-1000rpm at flow rates of 0.25-0.5ml/min where the partition efficiency of several 100 theoretical plates was achieved with over 50% stationary phase retention.     

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.     

Recent advances in counter-current chromatography

During the past several years, counter-current chromatography (CCC) technology has been advanced to cover a broad spectrum of applications, from large-scale preparative to analytical-scale separations. These advances include liquid-liquid dual CCC, foam CCC and partition of macromolecules with aqueous-aqueous polymer phase systems. For these developments the synchronous coil planet centrifuge scheme has been used, which relies on a relatively simple mechanical design. Future developments in CCC may be focused on the improvement of the more intricate non-synchronous coil planet centrifuge scheme which has a greater potential for the separation of biopolymers and cell particles.     

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.     

Protein separation with aqueous-aqueous polymer systems by two types of counter-current chromatographs.

Two different types of counter-current chromatographs, the cross-axis coil planet centrifuge (X-axis CPC) and horizontal flow-through coil planet centrifuge (horizontal CPC), were evaluated for protein separation with an aqueous-aqueous two-phase polymer system. The sample solution, containing 10-200 mg each of cytochrome c, myoglobin, ovalbumin and hemoglobin in 2 ml of each phase was eluted with the lower phase. In both instruments, the effects of the flow-rate, revolution speed, and parameter beta (helical diameters of the multilayer coil) on the protein separation were investigated. The best results were obtained from the X-axis CPC operated at 750 rpm and a flow-rate of 2.0 ml/min using a multilayer coil with a small helical diameter (beta = 0.25-0.60). Four protein samples were well resolved in less than 5 h.     

Performance of an aqueous two‐phase‐based countercurrent chromatographic system for horseradish peroxidase purification

Countercurrent chromatography (CCC) purification of horseradish peroxidase (HRP) from Armoracia rusticana root extracts was achieved by employing polymer‐phosphate aqueous two‐phase systems (ATPS). By using preparative columns at 1000 rpm, a 25–30% retention of the top phase of an ATPS composed of 10% w/w PEG 1540 and 14.8% w/w phosphate – with added 2 mol/kg sodium chloride – was obtained. The retention level was stable during the standard separation running time (4 h). Horseradish root extract samples were injected into the system (10–25 mL; 200–250 U/mL peroxidase; 2.0–4.0 mg/mL total protein). Retention of HRP in the CCC “column” during the chromatographic run was attained in the selected ATPS, where the partition coefficient K for the enzyme was ≥ 8. Replacement of the mobile phase with a fresh one but in the absence of added salt brought about product elution. Recovery of HRP in this fraction accounts for ≥ 45% of the total activity loaded, with a purification factor of 6. Enzyme activity was also found in the pass‐through fraction and in the remaining liquid (stationary) phase, a fact that should be ascribed to the existence of multiple peroxidase isoforms. SDS‐PAGE of the active fraction showed a protein band at 44 kDa, compatible with the presence of HRP. Thus, the optimised CCC system allowed the separation of HRP directly from a complex biological material. These results open up the possibility of achieving protein separation with CCC/ATPS and of scaling‐up processes in industrial separators.     

高速逆流色谱双水相体系分离蛋白质

利用多分离柱高速逆流色谱仪,研究了聚乙二醇1000(PEG1000)-磷酸盐双水相体系的固定相保留率及该体系对蛋白质混合物和鸡蛋清样品的分离。以14.0%PEG1000-16.0%磷酸盐体系的上相为固定相,在流速0.6 mL/min和转速900 r/min的条件下,固定相的保留率达到33.3%。在pH 9.2的PEG1000-磷酸盐双水相体系中,细胞色素C、溶菌酶和血红蛋白的分配系数差异最大,采用该pH值的14.0%PEG1000-16.0%磷酸钾盐双水相体系,在流速1.0 mL/min和转速850 r/min的条件下,成功地分离了这3种蛋白质的混合物。鸡蛋清中的主要蛋白质成分卵转铁蛋白、卵白蛋白和溶菌酶在pH 9.2的15.0%PEG1000-17.0%磷酸钾盐体系中也具有最大的分配系数差异。采用该体系,在流速1.0 mL/min和转速850 r/min的条件下,成功地分离了鸡蛋清样品,得到的卵白蛋白、溶菌酶和卵转铁蛋白的电泳纯度分别为100％,100％和60％,收率均大于90％。     

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.     

Tubing modifications for countercurrent chromatography: Investigation of geometrical parameters

Countercurrent chromatography (CCC) is a separation technique which may be described as a combination of a great number of liquid–liquid distributions of analytes in a two-phase solvent system with liquid chromatography (LC) features. Even optimized CCC separations currently provide a lower number of theoretical plates when compared to LC. For this reason, instrumental advancements are indispensable to, at least partly, overcome this drawback. Recently, we found that improvement of the classic CCC coil, that is using a long hollow tubing, may be achieved by the introduction of tubing crimpings which increase the stationary phase retention. In this study, we systematically investigated the effects of three geometrical parameters (crimping depth, distance between two crimpings as well as partial or complete crimping of the tubing) on the stationary phase retention by a factorial design of experiments approach. Separation efficiency tests were performed with two groups of analytes: fatty acid methyl esters (FAME) in the n-hexane/acetonitrile (HAcn) and alkyl p-hydroxybenzoates in the n-hexane/tert-butylmethylether/methanol/water solvent system. The most narrow crimping distance and the deepest crimping of the tubing were the best configurations in the examined flow rate range.     

Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems

Dual high-speed countercurrent chromatography (dual CCC) literally permits countercurrent flow of two immiscible solvent phases continuously through the coiled column for separation of solutes according to their partition coefficients. Application of this technique has been successfully demonstrated by separation of analytes by gas–liquid and liquid–liquid two-phase systems. However, the method cannot be directly applied to the system with a set of coiled columns connected in series, since the countercurrent process is interrupted at the junction between the columns. However, this problem can be solved by intermittent dual CCC by eluting each phase alternately through the opposite ends of the separation column. This mode of application has an advantage over the conventional dual CCC in that the method can be applied to all types of CCC systems including hydrostatic equilibrium systems such as toroidal coil CCC and centrifugal partition chromatography. Recently, the application of this method to high-speed CCC (hydrodynamic system) has been demonstrated for separation of natural products by Hewitson et al. using a set of conventional multilayer coil separation columns connected in series. Here, we have developed a mathematical model for this intermittent dual CCC system to predict retention time of the analytes, and using a simplified model system the validity of the model is justified by a series of basic studies on both hydrodynamic and hydrostatic CCC systems with a computer-programmed single sliding valve. The present method has been successfully applied to spiral tube assembly high-speed CCC (hydrodynamic system) and toroidal coil CCC (hydrostatic system) for separation of DNP-amino acid samples with two biphasic solvent systems composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1 and 4:5:4:5, v/v).     

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%.     

Gram Quantity Separation of DNP (Dinitrophenyl) Amino Acids with Multi-Layer Coil Countercurrent Chromatography (CCC)

Abstract

The efforts have been successfully made to extend the preparative capability of the high-speed CCC scheme with a multi-layer large capacity coiled column. The apparatus is a table top model of a horizontal flow-through coil planet centrifuge which produces a synchronous planetary motion of the column holder. The separation column was prepared from a single piece of 70 m long, 2.6 mm i.d., PTFE tubing coiled around the spool-shaped holder to form multiple layers of the coil with a total capacity of about 400 ml. The performance of the apparatus was assessed with a standard set of DNP amino acid samples and a two-phase solvent system composed of chloroform, acetic acid and 0.1N HCl (2:2:1). Preparative capability of the method was evaluated in terms of the retention level of the stationary phase and peak resolution for various sample size ranging from 0.05g to 2g. The effects of sample volume, sample concentration and the choice of the sample diluent on the separation were studied. The results indicated that both the retention level and the peak resolution tend to decrease with the increase of the sample volume applied at a given concentration. For separation of 1 gram quantity, best results were obtained by applying the sample dissolved in a small volume (10 ml) consisting of equal amounts of the two phases. Overall results indicate that the present scheme is capable of efficient separation for gram quantity of samples in a short period of time. The preparative capability may be further increased by the use of a larger-diameter and/or longer coil.     

Service life of counter-current chromatography coils

A multilayer coil of PTFE tubing, which failed after being used each workday for about 3 years in a type J centrifuge, was examined. Two types of defects were found. One, called crazes, occurs throughout the coil and does not leak initially, but may eventually lead to a short, axially oriented slit. Another, called indentations, is seen primarily in the innermost and other nearby layers. They are elongated, about 5 mm, indentations, usually on the central side of the tubing. These eventually crack and leak. PTFE tubing is permeable to air and hexane and expands by more than 1% when immersed in hexane, heptane or chloroform for a few days. It is suggested that the crazes result from exposure of the somewhat flexible tubing to the undulating centripetal force field in the coil-planet centrifuge, especially when further softened by solvent absorption. The indentations may result from carriage of the excess tubing length, created by solvent absorption, from the coil periphery to the coil center by the centripetal force field, which continuously travels from the peripheral tail to the central head of the coil. A 1% increase in coil length creates 74 cm of excess tubing in the 160-ml coils examined in this study. It is suggested that fluorinated ethylene propylene (FEP) tubing, especially when etched on the outside, may provide more stable CCC coils, since its expansion when exposed to organic solvents is 0.1 or less than that of PTFE.     

Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography

A spiral disk assembly composed of five single-channel units was designed for high-speed counter-current chromatography (HSCCC). The retention of different solvent systems ranging from moderately polar to polar organic-aqueous systems to aqueous two-phase systems (ATPs) was investigated under different elution modes. The results indicated that the spiral disk assembly can produce excellent retention of stationary phase for moderately polar organic-aqueous solvent systems, such as chloroform-methanol-water (4:3:2) and hexane-ethyl acetate-methanol-water (1:1:1:1) by pumping lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a high flow-rate (8 mL/min, Sf>70%), regardless of whether the inlet is at the inner or outer terminal of the channel. This makes it possible for fast analysis of some small molecular compounds. This has been proved in the separation of mixtures of three flavones, including isorhamnetin, kaempferol, and quercetin. The spiral disk assembly can also provide satisfactory retention for polar to ATPS such as 1-butanol-acetic acid-water (4:1:5) (<3 mL/min, Sf>70%), 12.5% poly(ethylene glycol) (PEG) 1000-12.5% K2HPO4-75% water (< or =1 mL/min, Sf>70%) and 4% PEG 8000-5% Dextran T500-91% water (< or =0.5 mL/min, Sf>50%) by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a low flow-rate, while this is not possible with the multilayer coil column. Acceptable resolutions were achieved when it was used for the separation of peptides such as Leu-Tyr and Val-Tyr, and proteins including cytochrome c and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins.