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Countercurrent chromatography (CCC) is an efficient separation technique without the solid support matrix, largely depending on the partition of two-immiscible liquid phases in the separation column. Since the helical coil planet centrifuge was invented in early 1970s by Yoichiro Ito, a series of coils columns, including spiral coils and conical coils columns have been developed for CCC separation. In this work, we introduced a new simple and efficient concentrical coils column for CCC separation, which was prepared by winding the whole polytetrafluoroethylene (PTFE) tube into the circular grooves from the rotation axis in the same direction. Once the PTFE tube filled in all space of one round of the circular groove, it was jumped into the nearby outer circular groove through the gap and until the whole groove was filled. The three same concentrical coils distributed on three disc-shaped holders were connected by the same PTFE tube to form concentrical coils separation column. The separation capacity was further investigated using ten tanshinones of the extracts of a Traditional Chinese Medicine Salvia miltiorrhiza Bunge as a model natural product. All results indicated that the concentrical coils column could hold satisfactory retention of the stationary phase, higher theoretical plate number and better resolution for CCC separation of more than ten tanshinones. It may be an alternative CCC column for non-targeted and targeted isolation of bioactive natural products.

Coil satellite centrifuge (CSC) produces the complex satellite motion consisting of the triplicate rotation of the coiled column around three axes including the sun axis (the angular velocity, ω1), the planet axis (ω2) and the satellite axis (the central axis of the column) (ω3) according to the following formula: ω1=ω2+ω3. Improved peak resolution in the separation of 4-methylumbelliferyl sugar derivatives was achieved using the conventional multilayer coiled columns with ethyl acetate/1-butanol/water (3: 2: 5, v/v) for the lower mobile phase at the combination of the rotation speeds (ω1, ω2, ω3)=(300, 150, 150rpm), and (1:4:5, v/v) for the upper mobile phase at (300:100:200rpm). The effect of the satellite motion on the peak resolution and the stationary phase retention was evaluated by each CSC separation with the different rotation speeds of ω2 and ω3 under the constant revolution speed at ω1=300rpm. With the lower mobile phase, almost constant peak resolution and stationary phase retention were yielded regardless of the change of ω2 and ω3, while with the upper mobile phase these two values were sensitively varied according to the different combination of ω2 and ω3. For example, when ω2=147 or 200rpm is used, no stationary phase was retained in the coiled column while ω2=150rpm could retain enough volume of stationary phase for separation. On the other hand, the combined rotation speeds at (ω1, ω2, ω3)=(300, 300, 0rpm) or (300, 0, 300rpm) produced insufficient peak resolution regardless of the choice of the mobile phase apparently due to the lack of rotation speed except at (300, 0, 300rpm) with the upper mobile phase. At lower rotation speed of ω1=300rpm, better peak resolution and stationary phase retention were obtained by the satellite motion (ω3) than by the planetary motion (ω2), or ω3>ω2. The effect of the hydrophobicity of the two-phase solvent systems on the stationary phase retention was further examined using the n-hexane/ethyl acetate/1-butanol/methanol/water system at different volume ratios. In the satellite motion at (ω1, ω2, ω3)=(300, 150, 150rpm), almost constant stationary phase retention was obtained with the lower mobile phase regardless of the hydrophobicity of the solvent system whereas the stationary phase retention varied according to the volume ratio of the two-phase solvent system for the upper mobile phase. However, stable stationary phase retention was observed with either phase used as the mobile phase. In order to analyze the acceleration acting on the coiled column, an acceleration sensor was set on the column holder by displacing the multilayer column. The combination of the rotation speeds at (300, 100, 200rpm) showed double loops in the acceleration track, whereas (300, 150, 150rpm) showed a single loop, and all other combinations showed, complex tracks. The overall results indicate that the satellite motion is seriously affected by the combination of rotation speeds and the hydrophobicity of the two-phase solvent system when the upper phase was used as the mobile phase for separation.

Separation of enantiomers still remains a challenge due to their identical physical and chemical properties in a chiral environment, and the research on specific chiral selector along with separation techniques continues to be conducted to resolve individual enantiomers. In our laboratory the promising magnetic chiral microspheres Fe3O4@SiO2@cellulose-2, 3-bis (3, 5-dimethylphenylcarbamate) have been developed to facilitate the resolution using both its magnetic property and chiral recognition ability. In our present studies this magnetic chiral selector was first purified by centrifuge field flow fractionation, and then used to separate benzoin racemate by a chromatographic method. Uniform-sized and masking-impurity-removed magnetic chiral selector was first obtained by field flow fractionation with ethanol through a spiral column mounted on the type-J planetary centrifuge, and using the purified magnetic chiral selector, the final chromatographic separation of benzoin racemate was successfully performed by eluting with ethanol through a coiled tube (wound around the cylindrical magnet to retain the magnetic chiral selector as a stationary phase) submerged in dry ice. In addition, an external magnetic field facilitates the recycling of the magnetic chiral selector.

Modern counter-current chromatography (CCC) originated from the helical coil planet centrifuge. Recently, spiral coils were found to possess higher separation efficiency in both the retention of stationary phase and solutes resolution than other CCC coils like the helical and toroidal coils used on type-J CCC and cross-axis CCC. In this work, we built a novel conical coil CCC for the preparative isolation and purification of tanshinones from Salvia miltiorrhiza Bunge. The conical coils were wound on three identical upright tapered holders in head-to-tail and left-handed direction and connected in series. Compared with helical and spiral coil CCC, conical coil CCC not only placed CCC column in a two-dimensional centrifugal field, but also provided a potential centrifugal force gradient both in axial and radial directions. The extra centrifugal gradient made mobile phase move faster and enabled CCC much higher retention of stationary phase and better resolution. As a result, higher efficiency has been obtained with the solvent system of hexane–ethyl acetate–methanol–water (HEMWat) with the volume ratio of 5:5:7:3 by using conical coil CCC apparatus. Four tanshinones, including cryptotanshinone ( 1 ), tanshinone I ( 2 ), 1,2-dihydrotanshinquinone ( 3 ) and tanshinone IIA ( 4 ), were well resolved from 500 mg to 1 g crude samples with high purity. Furthermore, the conical coil CCC can make a much higher solid phase retention, which makes it to be a powerful separation tool with high throughput. This is the first report about conical coil CCC for separation of tanshinones and it may also be an important advancement for natural products isolation.

Highlights ► Phase behaviour of an aqueous two-phase system was visualised in counter-current chromatography. ► High density difference causes phase bilateral distribution anomalies. ► Low interfacial tension or settling time makes columns not retain stationary phase. ► Mobile phase flow orientation rule is modified., This paper has addressed decade sought-after questions on phase bilateral distribution and stationary phase retention in any J-type high-speed counter-current chromatographic (CCC) centrifuge. Using a 2-D spiral column operated on such a CCC device and an aqueous two-phase system, this work systematically observed the phase interaction during transitional period and at dynamic equilibration under stroboscopic illumination. The experimental results thus obtained were used to examine the effects of the liquid–solid friction force, tangential centrifugal force, and physical properties of the two-phase system on hydrodynamic phase behaviour. We identified that (a) density difference between lower and upper phases is the critical factor to cause unusual phase bilateral distribution in the 2-D spiral column and (b) interfacial tension (manifested primarily as phase settling time) of any two-phase system is the critical factor in explaining inability to retain stationary phase in 3-D helical column and, for certain flow modes, in the 2-D spiral column. This work thus has extended or modified the well-established rule-of-thumb for operating J-type CCC devices and our conclusions can accommodate virtually all the anomalies concerning both hydrophobic and hydrophilic phase systems. To this end, this work has not only documented valuable experimental evidences for directly observing phase behaviour in a CCC column, but also finally resolved fundamentally vital issues on bilateral phase distribution orientation and stationary phase retention in 2-D spiral and 3-D helical CCC columns. Revised recommendations to end users of this technology could thus be derived out of the essence of the present work presumably following further experimental validation and a consensus in the CCC R&D and manufacturing circle.

Type-I coil planet centrifuge produces a uniformly circulating centrifugal force field to produce vortex motion of two immiscible solvent phases in a cylindrical cavity of the separation column to perform efficient countercurrent chromatography. The partition efficiency obtained from the original vortex column was substantially improved by threading the cylindrical cavity to increase the area of mass transfer between the two phases. Partition efficiency of the threaded column was evaluated by three different two-phase solvent systems with a broad range of hydrophobicity each with a set of suitable test samples. Overall results of the present studies indicated that the threaded cylindrical column substantially improves the partition efficiency in terms of theoretical plate number, peak resolution, and height equivalent of one theoretical plate. The results also indicated that higher peak resolution is produced by eluting either the upper phase in the head to tail direction or the lower phase in the reversed direction. When there is a choice in the mobile phase, a better separation is achieved by using the less viscous phase as the mobile phase. Since the present system gives extremely low column pressure, it may be a potential alternative to the conventional type-J HSCCC system for a large-scale preparative separation.

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-0.1. M 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).

I-type, J-type and non-synchronous centrifuges are all coil planet centrifuges. Analysing the motion of I-type and J-type centrifuges has advanced the understanding of how to manufacture and use these centrifuges. This paper analyses the motion of non-synchronous centrifuges producing equations of motion that can be applied to all coil planet centrifuges. This has also produced simple equations to determine the critical beta-values for any coil planet centrifuge. This paper also demonstrates that I-type centrifuges also have 2 critical beta-values when it was thought that beta-value did not influence the understanding of the processes within I-type centrifuges. For the I-type instrument both of these critical values are at bobbin radii approaching infinity. In practice this means all I-types function within one beta-value range hence the unilateral distribution and type/effectiveness of the mixing is consistent. Finally the paper shows the influence that the tangential velocity has on the Archimedean screw effect and thus the unilateral distribution of the upper and lower phases in the columns of coil planet centrifuges. This explains why the maximum stationary phase retention in an I-type centrifuge is limited to 50%.

Both batch and continuous separations were performed on an industrial liquor using a specially built continuous counter-current extraction centrifuge. Changing the flow regime for different batch separations showed that the elution of components from the respective ends of the coil depends on the flow rates of both upper and lower phases. It was shown that, within the scope of the study, the elution of the components was not affected by the concentration of the injected reaction liquor and more importantly that continuous processing with a counter-current chromatography centrifuge was feasible. This research represents an important step forward in making continuous counter-current chromatography (or true moving bed chromatography) accessible for the pharmaceutical industry.

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.

Synthetic peptides with many aromatic, aliphatic and especially acidic amino acid residues are not very soluble and require strong solvents for useful partitioning. A chloroform-methanol-acidic solvent system fractionates neutral and basically charged 26-mers to provide high yields. An insoluble 15-mer with 5 Trp residues and 60% overall hydrophobic amino acid content was purified in pyridine-acetic acid and in another basic t-butyl methyl ether-n-butanol-acetonitrile solvent system with high recovery. Two instruments were used, the eccentric-multi-layer hybrid coil planet centrifuge and the new spiral disk planet centrifuge that were able to retain the stationary phase of these solvent systems, some of which have low interfacial tension.

The cross-axis coil planet centrifuge (X-axis CPC) is useful for partitioning macromolecules with aqueous–aqueous polymer phase systems. The floor model we have built with a pair of separation columns had some shortcomings such as requirement of large space, short life of the flow tubes, and difficulty in installing columns. In order to improve the partition efficiency and the utility of counter-current chromatography (CCC), a new small-scale X-axis CPC was designed and fabricated in our laboratory. The down-sizing of the apparatus was done by reducing the scale to about 1/2 of our original model of X-1.5 L type with several improvements. Performance of the apparatus was evaluated on protein separation using an aqueous–aqueous polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate with four multilayer coiled columns. A series of experiments revealed that the combination of right- and left-handed coils produced the best partition efficiencies for both lower and upper mobile phases by selecting the revolution direction. The overall results indicate that the head–tail elution mode substantially affects to the peak resolution and stationary phase retention. This new X-axis CPC would be useful for the separation of various kinds of biologically active compounds.

Preparative counter-current chromatography (CCC) purification of valrubicin (AD-32) from crude synthetic drug has been successfully performed for the first time using upright coil planet centrifuge with four multilayer coils connected in series with 1600 ml capacity. The two-phase system used was composed of light petroleum (bp 60–90 °C)–ethyl acetate–tetrachloromethane–methanol–water at an optimized volume ratio of 1:1:8:6:1. Target compound (1.2 g) with a purity of 99.88% was obtained from 1.5 g of crude synthetic drug with a purity 95.49% based on HPLC peak area percentage. Identification of the target compound was performed by electrospray ionization mass spectrometry, one- and two-dimensional nuclear magnetic resonance.

Countercurrent chromatography (CCC) is a liquid chromatography (LC) technique with a special column able to retain a liquid stationary phase while the liquid mobile phase is pumped through. The coil planet centrifuge machines are made of open tube wound on spools. A simple test is proposed. The methanol–water (90:10, v/v)–heptane biphasic system is used with heptane as the mobile phase in the ascending or tail-to-head mode. The methanol–water stationary phase retention volume is measured at different flow-rates and rotor rotation speeds. After every machine equilibration, an alkylbenzene mixture is injected and the retention factors, peak efficiencies and resolution factors are measured or calculated for each solute. The wealth of information contained in the data set obtained is demonstrated. Four coil planet centrifuge machines of very different characteristics and one hydrostatic CCC machine with channels and ducts were submitted to the test. It was shown that the Sf, stationary retention factor, obtained with these machines was linearly dependent on the square root of F, the mobile phase flow-rate [Q. Du, C. Wu, G. Qian, P. Wu, Y. Ito, J. Chromatogr. A 835 (1999) 231–235]. It is shown that the slopes of the Sf versus F1/2 lines could be related to a minimum rotor rotation, ωmini, necessary to obtain the hydrodynamic equilibrium. The Sf and F parameters give the mobile phase linear velocity, u. It is shown that u is proportional to the square root of ω, the rotor rotation speed. The slope and intercept of the latter relationship also result in an ωmini value coherent with the first one. With the peak efficiencies and chromatographic resolution factors obtained for toluene and hexylbenzene, the parameters: number of plates per tubing turn, machine volume for one plate, and tubing length for one plate, were calculated and compared for the five machines. The internal diameter of the tubing used is shown to be a critical parameter acting on the machine volume and number of tubing turns.

This paper deals with optimization of a new seal-free compact toroidal coil centrifuge to achieve high resolution in analytical counter-current chromatography (CCC). Toroidal coil CCC (hydrostatic motion) has advantages compared with high-speed CCC (efficiently mixing solution with planetary motion) in the separation of protein or easily emulsified samples. A toroidal coil separation column of 0.4 mm I.D. PTFE tubing was accommodated around the periphery of the cylindrical centrifuge bowl. Using a two-phase solvent system composed of chloroform–acetic acid–0.1 M hydrochloric acid (2:2:1, v/v/v) and a set of dinitrophenyl-amino acids as test samples, a series of experiments was performed with parameters such as the column length, sample volume, flow-rate, elution mode of the mobile phase and rotation speed. The highest efficiency, over 10 000 theoretical plates, was achieved with a 100 m long coiled tube and an 11 ml total capacity at a flow-rate of 0.01 ml/min at 800 rpm.

The present studies have been conducted to investigate the cause of an unusually broad peak of ovalbumin obtained by countercurrent chromatography (CCC) reported earlier [K. Shinomija et al., J. Chromatogr., 644 (1993) 215]. A series of CCC experiments using our prototype of the cross-axis coil planet centrifuge revealed that commercial ovalbumin products were classified into two groups: group A formed two peaks of ovalbumin at pH 7.0 and 5.8, while group B showed a relatively sharp single peak in a broad range of pH. Electrophoresis indicated that the group A ovalbumin consisted of both natural and denatured products: the natural ovalbumin is a monomer ( M r 45 000) whereas the denatured products form dimers ( M r 90 000). The abnormally broad peak obtained from the group A ovalbumin at pH 9 is apparently caused by the heterogeneity of the sample protein.

Preparative separations of peptides have been accomplished using high-speed counter-current chromatography. This has been made possible by the use of a particular solvent system that does not exhibit solvent carryover at high speed flow and centrifugation conditions. The solvent system composed of tert.-butyl methyl ether, n-butanol, acetonitrile and aqueous trifluoroacetic acid which can be adjusted in volume ratios and percent acid is employed in two instruments for counter-current chromatography. The cross-axis coil planet centrifuge was used for separation of various dipeptides. Superior resolution was obtained with the multi-layer coil planet centrifuge in the separation of six dipeptides and in the preparative purification of two synthetic peptides.

A new counter-current chromatograph, named the cross-axis coil planet centrifuge, has been optimized through various versions since 1987. Features of this recently commercialized apparatus are reviewed. It allows the use of biphasic polar solvent systems, of aqueous solutions of polymers and systems containing an organic phase of small or moderate hydrophobicity. Separations and purifications achieved on the cross-axis device demonstrate that it provides a good efficiency and resolution with very high retention of the stationary phase at convenient flow-rates, whatever the solvent system used.

Retention of the stationary phases of one organic-aqueous solvent system and three aqueous-aqueous polymer solvent systems was investigated on a cross-axis coil planet centrifuge. A graphic statistical treatment of all the data highlighted the prevailing effect of the inward-outward elution mode. A simplified model was proposed and studies on the paths and accelerations of cross-axis devices described in the previous paper provided explanations about the observed hydrodynamic behaviors.

A comprehensive approach is introduced using parametric equations to describe the motion induced by type J and cross-axis type coil planet centrifuges. The plots of a line parallel to the coil axis are given. The centrifugal forces are then analyzed in two ways. Three-dimensional graphs show their geometry and the relative intensity of the lateral component is compared to that of the perpendicular component. This allowed a better understanding of the differences in paths and acceleration fields induced by the two types of counter- current chromatography devices.

An improved model of the cross-axis synchronous flow-through coil planet centrifuge has been designed in light of previous studies. The apparatus has a versatile feature in that both analytical and preparative columns can be accommodated in both off-center and central positions. Each has merit in separations. Retention of stationary phase was examined with various two-phase solvent systems used for the separation of biopolymers. Both analytical and preparative columns showed satisfactory retention of the stationary phase under optimum conditions. The apparatus was evaluated in separation of a set of protein samples using a polyethylene glycol—potassium phosphate biphasic system. In both types of columns all proteins were resolved with partition efficiencies of 260 to 670 theoretical plates. Further studies indicated that the relatively low partition efficiency of proteins is mainly attributed to their high molecular mass or molecular heterogeneity within each species rather than due to the high viscosity of the polymer phase system.

A general introduction is given to modern countercurrent chromatography, high-speed countercurrent chromatography using the coil planet centrifuge and centrifugal droplet countercurrent chromatography (centrifugal partition chromatography). The two techniques offer new capabilities for the retention of the liquid stationary phase in the column via a strong centrifugal field and for increased efficiency. Thus, the flow-rate of the mobile phase permits faster separations in comparison with previous techniques such as droplet countercurrent chromatography or rotation locular countercurrent chromatography. Examples are given to show that this is a powerful technique for the preparative-scale separation of natural compounds and it can be coupled to various detection system.

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.

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.

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.

Horizontal flow-through coil planet centrifuge (CPC) and high-performance liquid chromatography (HPLC) techniques were used for separation of polar compounds from a crude ethanol extract of Oxytropis ochrocephala Bunge, a poisonous legume plant widely distributed in northwestern China. The performance of these two chromatographic methods was compared in terms of column efficiency, peak resolution, separation time, sample loading capacity, etc. The results indicated that two polar compounds in the crude extract were equally well separated by these two methods. HPLC gave comparable peak resolution in shorter separation time while its sample loading capacity was limited to the mg range. The CPC method required a long separation time, but yielded a higher purity of fractions with a much greater capacity.

In order to further extend the preparative capability of the apparatus, we have constructed a new centrifuge which can hold much longer multilayer coils for the separation of multigram quantities of samples. The performance of the apparatus has been successfully demonstrated in the separation of dinitrophenyl (DNP) amino acids using a two-phase solvent system composed of chloroform-acetic acid 0.1 M hydrochloric acid at a volume ratio of 2:2:1

Counter-current chromatography (CCC0 is mainly used in the preparative separation, extraction and purification of samples. CCC does not operate with a solid stationary phase. Two immiscible liquid phases are used. One is the liquid mobile phase and the other is the liquid stationary phase. Centrifugal fields are used to retain the liquid stationary phase while the mobile phase is pushed through it. The CCC “column” if often a continuous open tube, coiled on a spool which is rotated in a centrifuge. Most high-speed counter-current chromatographs are scheme IV Ito coil planet centrifuges. A practical CCC approach is reported in this paper. The user-adjustable parameters are divided into (i) configuration parameters and (ii) operating parameters. The configuration parameters are the tube internal diameter, the spool radius, the number of turns and the total internal apparatus volume. The operating parameters are three active parameters (mobile phase flow-rate, spool rotation speed and temperature) and two passive parameters (the stationary phase retention percentage and the driving pressure). The choice of solvents to be used as the biphasic liquid systems is critical. Tie lines in ternary phase diagrams are used to optimize the solvent choice with hexane—methanol—water and the chloroform—methanol—water systems. The effects of interdependent parameters on the CCC chromatograms are discussed and illustrated with examples. It is shown that a highly efficient counter-current chromatograph can have a poor resolving power if it cannot retain a sufficient amount of liquid 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

Diaziquone is an active antitumor agent in animals that is unstable in aqueous solution. The chemical hydrolysis products of this antitumor agent have been identified and we have investigated the usefulness of the coil planet centrifuge in separating these products from the parent compound. We were able to separate the reactive diaziquone from the two reaction products using a solvent system containing dichloroethane, methanol, and sodium phosphate buffer. The identities of the products separated on the coil planet centrifuge were confirmed by thin-layer chromatography and mass spectrometry. This technique may be useful in separating metabolites contained in specimens obtained from diaziquone-treated patients.

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.

Several schemes are presented which illustrate the general utility of column chromatography on diol-bonded silica gel for the purification of various antibiotics. The antibiotics include rosarimicin, rosarimicin dimethylacetal, everninomicin D, Juvenimicin A4, coloradocin and the benzanthrins. A set of partition coefficients, determined in different two-phase solvent systems, for a given antibiotic or antibiotic complex, can be used in selecting appropriate solvent systems for this chromatography as well as for semi-preparative counter-current chromatography on the Ito coil planet centrifuge.

Extensive studies have been performed on hydrodynamic distribution of two immiscible solvent phases in helical and spiral columns by means of coil planet centrifugation. Under the Scheme IV [see Fig. 1 in Y. Ito, J. Chromatogr., 301 (1984) 377] planetary motion, the two phases display unilateral distribution in the coil, i,e., one phase travels toward the head and the other phase toward the tail. This hydrodynamic trend is sensitively affected by various factors such as physical properties of the solvent system, helical diameter of the column, revolutional radius, etc. Phase distribution diagrams obtained from a set of conventional two-phase solvent systems under various experimental conditions provide extremely useful information for both basic hydrodynamic studies and practical application in high-speed counter-current chromatography.

A new angle rotor coil centrifuge (Ψ = 25°), 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 acids samples with chloroform—acetic acid—0.1 N hydrochloric acid (2:2:1).

A compact table-top model of a flow-through coil planet centrifuge produces and efficient chromatagraphic separation of solutes on a preparative scale within 2–5 h. The use of a multi-layer coiled column promotes retention of the stationary phase with a high flow-rate of the mobile phase and permits almost universal application of conventional two-phase solvent systems. The capability of the counter-current chromatography scheme has been demonstrated on separations of various biological samples, such as dinitrophenyl amino acids, dipeptides, gramicidins, auxins, purines and pyrimidines.

The new horizontal flow-through coil planet centrifuge allows continuous elution through a pair of coiled separation columns without the use of rotating seals. Design of the apparatus enables each column to undergo a specific mode of synchronous planetary motion which produces a charateristic pattern of the acceleration field as described in Part I. Using typical two-phase solvent systems, retention of the stationery phase and partition efficiency were studied for each column under various revolutional speeds and flow-rates. The results indicate that one column enables preparative-scale separations and the other, analytical-scale separations both with a high partition efficiency comparable to that obtained refined liquid chromatography.

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 centrfugal force field which promises high retention of the stationary phase in the multilayer coil to perform efficient preparative-scale counter-current chromatography.

A compact table-top model of the coil planet centrifuge simultaneously enables both preliminary purification and enrichment of samples from crude extracts or biological fluids. The method uses hydrodynamic behavior of two immiscible solvent phases in a rotating coiled tube to retain the stationary phase against a high flow-rate of mobile phase. Consequently, a small quantity of the sample present in a large volume of mobile phase is efficiently extracted into a small volume of the stationary phase within a short period of time and at a high recovery rate. The capability of the present method was demonstrated in the extraction of dinitrophenylamino acids (used as a comparative performace standard) with a set of two-phase solvent systms composed of ethyl acetate and 0.5 M NaH 2 PO 4 .

The new type of horizontal flow-through coil planet centrifuge enables both preparative and analytical separations with partition efficiencies comparable to liquid chromatography. This counter-current chromatographic method was evaluated by the separation and purification of DNP-amino acids and various kinds of peptides using common two-phase solvent systems. The peptide samples separated include commercially available oligopeptides and two synthetic peptides of five and fifteen amino acids. A partition efficiency of up to several thousand theoretical plates was obtained with DNP-amino acids. Purification of the synthetic peptides, which produced problems with conventional liquid chromatography due to contamination and poor solubility, was successfully performed on a preparative scale with a reasonable degree of recovery.

A countercurrent chromatography apparatus and method where the column rotates about an axis spaced apart from, parallel to, and in the same radial plane as a radius extending from the central axis of revolution. The apparatus generates a unique force field which enables excellent separation.

A rotary-seal-free planetary centrifuge holds a separation column which consists of multiple partition units (ca. 200) connected in series with transfer tubes. In the cavity of each partition unit the transfer tube extends to form a mixer which vibrates to stir the contents under an oscillating force field generated by the planetary motion of the centrifuge. Consequently, solutes locally introduced at the inlet of the column are subjected to an efficient partition process in each partition unit and separated according to their partition coefficients. The mixer tube equipped with a flexible silicone rubber joint was found to produce excellent results for partition with viscous polymer phase systems. The capability of the method was demonstrated on separation of cytochrome c and lysozyme using a PEG-aqueous dibasic potassium phosphate-aqueous two-phase solvent system.

A compact desktop model of a high-speed counter-current chromatograph holds three identical multilayer coils in the symmetrical positions around the rotary frame to maintain perfect balance of the centrifuge system without the use of a counterweight. These multilayer coils are connected in series to make up a total capacity of 400 ml while the unique gear arrangement on the rotary frame establishes a twist-free mechanism of the flow tubes so that continuous elution can be performed without the use of rotary seal. The high performance of the present system was successfully demonstrated in separations of 10–250 mg of 2,4-dinitrophenyl amino acid mixtures in a two-phase solvent system composed of chloroform—acetic acid—0.1 M hydrochloric acid (2:2:1, v/v/v).

The coil planet centrifuge designed by Ito employs flow of a single liquid phase, through a rotating coiled tube in a centrifugal force field, to provide a separation of particles based on sedimentation rates. Mathematical solutions are derived for the linear differential equations governing particle behavior in the coil planet centrifuge device. These solutions are then applied as the basis of a model for optimizing particle separations.

A horizontal flow-through coil planet centrifuge was constructed that holds a pair of coiled separation columns made of PTFE tubing of different inner diameters. With this scheme, samples from a few to hundreds of milligrams can be separated in each operation. The experimental methods are discussed from three aspects: the selection of the two-phase solvent systems, the determination of the optimal operational conditions and the general separation procedures. The capabilities of this apparatus were demonstrated with separations of several biological samples such as antibiotics, agricultural chemicals and medicinal herbs.

A method for chromatographic separation of macromolecules by the use of centrifugation through gel filtration materials is described. The liquid excluded from the gel material is removed by centrifugation; thus, this method offers rapid separation and minimal dilution of samples. Because of the rapid separations obtained, this method is particularly suitable for study of enzyme reactions, ligand binding and formation of intermolecular complexes in general. Within the limitations of the short columns which were employed in the centrifuge, the resolution of macromolecules is equivalent on columns eluted conventionally and columns eluted with the assistance of centrifugation.