Cross-axis synchronous flow-through coil planet centrifuge (type XLL). I. Design of the apparatus and studies on retention of stationary phase

The fourth prototype holds a pair of column holders in the lateral position at 15 cm from the center of the rotary shaft horizontally mounted on the rotary frame at 7.6 cm from the central axis of the apparatus. Using short coils of 2.6 mm I.D. PTFE (polytetrafluoroethylene) tubing with 7.6 cm and 24 cm helical diameters, retention of the stationary phase was measured in ten pairs of two-phase solvent systems under various experimental conditions. Satisfactory retention was obtained by choosing proper combinations of three factors, i.e., the direction of planetary motion, head-tail elution mode, and inward-outward elution mode. The polar butanol solvent systems showed excellent retention from 65 to 80% in the 7.6 cm helical diameter left-handed coil.     

Improved cross-axis synchronous flow-through coil planet centrifuge for performing counter-current chromatography. I. Design of the apparatus and analysis of acceleration

A novel design of the cross-axis synchronous flow-through coil planet centrifuge is introduced. The apparatus holds a pair of large coil holders symmetrically, one on each side of the rotary frame, at a lateral position 12.5 cm from the center of the holder shaft held 10 cm from the centrifuge axis. Mathematical analysis of acceleration generated by the planetary motion of the apparatus revealed a unique centrifugal force field which promises high retention of the stationary phase in the multilayer coil to perform efficient preparative-scale counter-current chromatography.     

Multilayer coil planet centrifuge for analytical high-speed counter-current chromatography

A compact portable model of a high-speed counter-current chromatograph enables efficient analytical separations of microgram sample quantities within 10 min. A series of preliminary experiments was conducted to study retention of the stationary phase of various two-phase solvent systems in short coils with different helical diameters. Under the optimal experimental conditions, analytical capability of the apparatus was successfully demonstrated in separation of flavonoids from a crude sea buckthorn ethanol extract in a multilayer coil with a total capacity of 8 ml.     

High-Speed Countercurrent Chromatography of Dipeptides on a Polar Two-Phase Solvent System

Abstract

The performance of the high-speed countercurrent chomatograph was evaluated by separating dipeptide samples on a polar biphasic solvent composed of n-butanol-acetic acid-water (4:1:5, v/v/v). Best results were obtained with a set of multilayer coils of small helical diameter by eluting with the upper nonaqueous phase in a head to tail direction at a flow rate of 1 or 2 ml/min. Four components were completely resolved in 5 to 11 hours. Other types of coiled columns such as a multilayer coil with large helical diameter (in a commercial model) and eccentric dual-layer coil assemblies mounted on a horizontal coil planet centrifuge also yielded satisfactory separations.     

Cross-axis synchronous flow-through coil planet centrifuge (type XLL). II. Speculation on the hydrodynamic mechanism in stationary phase retention

The hydrodynamic mechanism involved in the retention of stationary phase in the present x-axis coil planet centrifuge system is discussed. A statistical treatment of the retention data disclosed important clues such as the effect of the inward-outward elution mode, the close correlation between the planetary motion and the head-tail elution mode, and the superior retention capacity of the left-handed coils. The combined effects of the radial and lateral centrifugal force field derived from the mathematical analysis of acceleration acting on the coil provide an explanation for the phenomena.     

Studies on the performance of different coiled column configurations for compact type-I countercurrent chromatography

Three types of novel coiled column configurations, i.e. a triangular coiled column and elliptical coiled columns I and II, were designed for type-I countercurrent chromatography and their performances were evaluated with two solvent systems each with suitable test samples. Three dinitrophenyl (DNP) amino acids (DNP-DL-glu, DNP-β-ala and DNP-L-ala) were separated with a moderately hydrophobic two-phase solvent system composed of hexane-ethyl acetate-metanol-0.1 M hydrochloric acid (1:1:1:1, v/v), while two dipeptides (tryptophyl-tyrosine and valyl-tyrosine) were separated with a polar solvent system composed of 1-butanol-acetic acid-water (4.75:0.25:5, v/v). The overall results indicated that the performance of compact type-I countercurrent chromatography was improved by elliptical coiled column I which was mounted with its maximum coil diameter perpendicular to the surface of the column holder. Hydrodynamic effects involved in these separations were discussed.     

Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor

An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid-liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane-ethyl acetate-methanol-water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol-potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor.     

Evaluation of the effect of column orientation in type-I high-speed counter-current chromatography

The performance of type-I high-speed counter-current chromatography was evaluated by changing the column inclination against the rotating centrifugal force field. The separations were performed with two different solvent systems composed of 1-butanol–acetic acid–water (4.75:0.25:5, v/v) (BAW) and hexane–ethyl acetate–methanol–0.1 M HCl (1:1:1:1, v/v) (HEMW) using dipeptides and DNP-amino acid as test samples, respectively. A set of short coiled columns connected in series is mounted around the holder hub in two different ways: in the parallel orientation, all column units are arranged in parallel to each other and mounted on the holder at various angles against the horizontal plane. In the zigzag configuration, the neighboring units of the same column are mounted symmetrically forming various angles apart. In the parallel configuration, for both the BAW and HEMW systems, Sf (the retention of stationary phase) first increased as the column angle decreased from 90° to 60° and then decreased, as the column angle further decreased from 60° to 0°, while Rs (peak resolution) continually declined over the entire column angle range from 90° to 0°. But, for both solvent systems, with the zigzag configuration, retention of stationary phase and resolution both decreased as the column angle decreased from 90° to 0°. In general, Sf and Rs for separation of dipeptides in the BAW system, from 90° to 15°, is better for the parallel orientation than for the zigzag configuration. However, at 0°, Sf and Rs are better for the zigzag orientation. In the DNP-amino acid separation with the HEMW system, retention of the stationary phase and Rs for the parallel orientation is better than that for the zigzag orientation from 90° to 30°, whereas from 30° to 0° the results are opposite. Over all results of our studies revealed that the formally used column orientation [5] at 90° inclination yields the highest peak resolution in both solvent systems.     

Experimental observations of the hydrodynamic behavior of solvent systems in high-speed counter-current chromatography. I. Hydrodynamic distribution of two solvent phases in a helical column subjected to two types of synchronous planetary motion

Hydrodynamic distribution of two-phase solvent systems in a rotating helical column subjected to centrifugal fields produced by two different types of synchronous planetary motion has been studied by the use of the combined horizontal flow-through coil planet centrifuge. With continuous elution of the mobile phase, the simpler type of motion resulted in low retention of the stationary phase in the column whereas a more complex motion, which produces a quasi-radial centrifugal field varying in both intensity and direction, yielded high stationary phase retention for commonly used solvent systems having a wide range of hydrophobicity. These solvent systems display highly complex modes of hydrodynamic interaction in the coil according to their particular physical properties.     

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.     

New angle rotor coil planet centrifuge for counter-current chromatography. II. Design of the apparatus and studies on phase retention and partition capability

A new angle rotor coil planet centrifuge (psi = 25 degrees), which produces a Type J-L synchronous planetary motion, has been constructed to examine its capability in terms of stationary phase retention and solute partitioning. Studies on phase distribution diagrams obtained from various two-phase solvent systems indicated that the present system can be adapted to a wide variety of solvent systems by adjusting the centrifugal conditions. Excellent partition capability of the apparatus was successfully demonstrated in separations of dinitrophenyl amino acid samples with chloroform-acetic acid-0.1 N hydrochloric acid (2:2:1).     

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.     

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.     

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.     

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.     

Preparative Purification of Peptides by Countercurrent Chromatography on the Ito Coil Planet Centrifuges

Abstract

For the purification of up to 1000 mg of synthetic peptides by countercurrent chromatography, the coil planet centrifuges have proven useful in the research laboratory. Besides the earlier described horizontal flow-through coil planet centrifuge which chromatographs substances in all solvent systems at room temperature, the multi-layer coil planet centrifuge affords more rapid chromatography. However, separations using n-butanol, especially suited for peptides, have to be conducted at elevated temperatures. A new machine that has characteristics of both of the foregoing instruments is the compact horizontal flow-through coil planet centrifuge which promises rapid chromatography with full retention of the stationary phase.     

Evaluation on the performance of four different column models mounted on the compact type-I coil planet centrifuge

Optimal positions of coiled separation columns on the type-I centrifuge were determined for four typical two-phase solvent systems to obtain the best separation efficiency (resolution and retention of stationary phase) for each with a suitable set of test samples. A set of short coiled columns is connected in series and mounted around the holder hub in four different ways: (model A) the tail of one unit with left-handedness was connected to the head of the next unit with right-handedness (TL-HR); (model B) the tail of one unit with left-handedness was connected to the tail of the next unit with right-handedness (TL-TR); (model C) the tail of one unit with left-handedness was connected to the tail of the next unit with left-handedness (TL-TL); (model D) the tail of one unit with left-handedness was connected to the head of the next unit with left-handedness (TL-HL). The results indicated that the performance of model D was the best among the four models. High revolution speed (800 rpm) is favorable to separation using the moderately hydrophobic solvent system of hexane-ethyl acetate-methanol-0.1M HCl (1:1:1:1, v/v) (HEMW), while lower revolution speed (600 rpm) is beneficial to the separation with polar solvent system of 1-butanol-acetic acid-water (19:1:20, v/v) (BAW).     

High-speed counter-current chromatography of apple procyanidins

Apple procyanidins were separated by high-speed counter-current chromatography using a type-J multilayer coil planet centrifuge. Several two-phase solvent systems with a wide range of hydrophobicities from a non-polar hexane system to polar n-butanol systems were evaluated their performance in terms of the partition coefficient and the retention of the phase. The best separation of procyanidins B and C was achieved with a two-phase solvent system composed of n-butanol-methyl tert.-butyl ether-acetonitrile-0.1% trifluoroacetic acid (2:4:3:8) using the lower phase as a mobile at a flow-rate of 1.0 ml/min.     

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.     

Optimization of a high speed countercurrent chromatograph for analytical separations

The analytical capability of the high speed countercurrent chromatograph model 4000 (HSCCC-4000) has been improved by optimizing the dimensions of the multilayer coiled column. Using a two-phase solvent system of n-hexane – ethyl acetate – methanol – water (1:1:1:1) and a set of indole auxins as test samples, a series of studies was conducted to evaluate performance of coiled columns with i.d.s ranging from 0.1 to 0.55 mm. The studies on the stationary phase retention indicated that multilayer coils of 0.55 mm and 0.30 mm i.d. provide satisfactory retention of the stationary phase while the 0.10 mm i.d. column fails to yield reproducible retention. The best partition efficiencies were obtained from the 0.30 mm i.d. multilayer coil with a 6 ml capacity which produced theoretical plate numbers ranging from 5500 to 10500 with a resolution factor of 2.30. The feasibility of interfacing the HSCCC-4000 with a mass spectrometer is briefly discussed.