Aqueous-aqueous Polymer Phase Systems Research Articles

Comparison in partition efficiency of protein separation between four different tubing modifications in spiral high-speed countercurrent chromatography.

High-speed countercurrent chromatography with a spiral tube assembly can retain a satisfactory amount of stationary phase of polymer phase systems used for protein separation. In order to improve the partition efficiency a simple tool to modify the tubing shapes was fabricated, and the following four different tubing modifications were made: intermittently pressed at 10 mm width, flat, flat-wave, and flat-twist. Partition efficiencies of the separation column made from these modified tubing were examined in protein separation with an aqueous-aqueous polymer phase system at flow rates of 1-2 ml/min under 800 rpm. The results indicated that the column with all modified tubing improved the partition efficiency at a flow rate of 1 ml/min, but at a higher flow rate of 2 ml/min the columns made of flattened tubing showed lowered partition efficiency apparently due to the loss of the retained stationary phase. Among all the modified columns, the column with intermittently pressed tubing gave the best peak resolution. It may be concluded that the intermittently pressed and flat-twist improve the partition efficiency in a semi-preparative separation while other modified tubing of flat and flat-wave configurations may be used for analytical separations with a low flow rate.

Improved Separations of Proteins and Sugar Derivatives Using the Small-Scale Cross-Axis Coil Planet Centrifuge with Locular Multilayer Coiled Columns.

Countercurrent chromatography (CCC) is liquid-liquid partition chromatography without using a solid support matrix. This technique requires further improvement of partition efficiency and shortening theseparation time. The locular multilayer coils modified with and without mixer glass beads were developed for the separation of proteins and 4-methylumbelliferyl (MU) sugar derivatives using the small-scale cross-axis coil planet centrifuge. Proteins were well separated from each other and the separation was improved at a low flow rate of the mobile phase. On the other hand, 4-MU sugar derivatives were sufficiently resolved with short separation time at a highflow rate of the mobile phase under satisfactory stationary phase retention. Effective separations were achieved using the locular multilayer coil for proteins with aqueous-aqueous polymer phase systems and for 4-MU sugar derivatives with organic-aqueous two-phase solvent systems by inserting a glass bead into each locule.

ChemInform Abstract: Chiral High‐Speed Counter‐Current Chromatography: Future Strategies for Chiral Selector Development

AbstractReview: 23 refs.

Chiral High-Speed Counter-Current Chromatography: Future Strategies for Chiral Selector Development

In conventional high-performance liquid chromatography, chiral separations are performed by chiral column with a chiral selector (CS) chemically boned to the solid support. In contrast, high-speed counter-current chromatography (HSCCC) performs chiral separations by dissolving CS in the liquid stationary phase. During the past two decades, several CSs were developed to successfully carry out chiral HSCCC which include N-dodecanoyl-L-proline-3,5-dimethylanilide, β-cyclodextrin derivatives, vancomycin, cinchona alkaloid derivatives, cellulose and amylose derivatives, tartaric acid derivatives, etc. Compared to HPLC which uses over hundred different kinds of CSs, the number of CSs effectively used in HSCCC is limited to several compounds. This may be due to the violent molecular movement of CS dissolved in the liquid stationary phase which reduces chiral selectivity based on steric affinity. Future development strategy of CS for HSCC proposed here is to suppress the molecular movement of the CS in the liquid stationary phase by the following three ways: 1) using viscous stationary phase such as aqueous-aqueous polymer phase system; 2) attaching a long hydrophobic chain to the asymmetric carbon, or 3) chemically bonding CS onto hydrophobic small particles such as carbon nanotubes, gold colloidal particles, and submicron silica particles.

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

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

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

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

PROTEIN SEPARATION BY CROSS-AXIS COIL PLANET CENTRIFUGE WITH SPIRAL COLUMN ASSEMBLIES

ABSTRACT A newly fabricated spiral column assembly was first applied to the countercurrent chromatographic separation of proteins using the cross-axis coil planet centrifuge (cross-axis CPC). The separation was performed by a set of stable proteins such as cytochrome C, myoglobin, and lysozyme with an aqueous–aqueous polymer phase solvent system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) dibasic potassium phosphate. Three sets of left-handed single-layer spiral columns with different IDs (1.0, 1.5, and 2.0 mm) were employed to investigate the effect of column ID on partition efficiency at four different elution modes. The elution modes consisted of the combinations of the direction of revolution (PI=counterclock-wise; PII=clockwise), the head–tail elution mode (H=head to tail; T=tail to head), and the inward–outward elution mode (I=inward; O=outward).   Among these experiments, the best separation of proteins was attained using a 1.5 mm-ID column assembly with the PI-H-O elution mode of the lower phase mobile. The resolution between cytochrome C and myoglobin peaks was 0.9 and between myoglobin and lysozyme peaks was 1.0, while the retention of the stationary phase was 32.7%. In order to improve the partition efficiency, the four-layer spiral column assembly with 1.0 mm-ID column tubing was applied to the protein separation. The resolution between these peaks was improved to 1.1 and 1.2, respectively, while the stationary phase retention decreased to 20.9%. Further studies using a single-layer spiral coiled column also revealed the effective peak resolution of proteins, but with lower stationary phase retention. The overall results demonstrated the spiral column assembly was useful for the protein separation using the cross-axis CPC with the aqueous-aqueous polymer phase system.

ONE-STEP PURIFICATION OF CHOLINESTERASE FROM HUMAN SERUM BY CCC

The type XL cross-axis coil planet centrifuge (CPC) was successfully utilized for purification of cholinesterase (ChE) from human serum by countercurrent chromatography (CCC). Aqueous-aqueous polymer phase systems composed of polyethylene glycol-potassium phosphate buffers were tested at various pHs, ranging from 6.8 to 9.2 for the distribution ratios (D) of ChE, human serum albumin, α- and γ-globulins. The CCC separation was performed with optimized polymer phase systems in two different coiled columns. The best purification was achieved with 16.0% PEG 600–12.5% potassium phosphate at pH 9.2, by eluting the lower phase at 0.5 mL/min through the small capacity column in which ChE was purified directly from human serum within 2h. The ChE fractions confirmed by the enzymatic activity were free of serum proteins by SDS PAGE analysis.

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

Proteins were separated using a cross-axis coil planet centrifuge equipped with two different types of coiled columns, i.e., toroidal and eccentric coil assemblies. An aqueous-aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate was used for the separation of cytochrome C, myoglobin and ovalbumin. The toroidal coil assemblies with 1 mm ID tubing yielded the partition efficiency of 170 theoretical plates at the myoglobin peak. A slightly higher efficiency was obtained from the eccentric coil assemblies with the same diameter. The highest partition efficiency of 450 theoretical plates was attained from the eccentric coil assemblies with 0.85 mm ID and 64 mL capacity.

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.

Polyethylene Glycol-Potassium Phosphate Aqueous Two-Phase Systems for Countercurrent Chromatography of Proteins

Aqueous-aqueous polymer phase systems composed of polyethylene glycol 1000-potassium phosphate were used for the countercurrent chromatographic separation of proteins using the type XL cross-axis coil planet centrifuge. It was found that the peak resolution of proteins is highly dependant on the pH of the solvent system. The best separation of cytochrome c, myoglobin and human serum albumin was achieved with 16.0% PEG 1000-12.5% potassium phosphate buffer at pH 9.2. The resolution and separation time were improved by pH-peak-focusing countercurrent chromatography.

Toroidal coil planet centrifuge for counter-current chromatography

A simple tabletop model of a versatile counter-current chromatographic system is introduced. The apparatus compactly holds a long coiled column around a drum-shaped holder. The acceleration produced by the synchronous planetary motion of the holder enables stable retention of the stationary phase in each helical turn of the coil while the mobile phase is continuously eluted through the column. The capability of the method was demonstrated on separations of DNP amino acids and oligopeptides using typical two-phase solvent systems. The present method will provide a universal application of solvent systems including aqueous-aqueous polymer phase systems used for partition of cell particles and macromolecules.