Improved partition efficiency with threaded cylindrical column in vortex counter-current chromatography

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.     

Effect of gravitational force on type-J counter-current chromatography by mathematical analysis

The gravitational force exerts an important effect on the counter-current chromatography (CCC) and has been used to retain the stationary phase and improve the efficiency in the various CCC separations. This paper deals with the effect of gravitational force on type-J CCC, one of the most popular CCC methods by the mathematical analysis for the first time. The theoretical analyses reveal a close relationship between the effect of gravitational force, the placed orientation of type-J CCC apparatus, and the revolutional speed. Two placed orientations are more suitable for preparative CCC separation: one is common horizontal, and the other upright. Thus, there are three optimum conditions for type-J CCC separation, the first using horizontal apparatus at very low revolutional speed around 10rpm in the gravitational filed, the second by use of upright apparatus at moderate revolutional speed around 100rpm in the three-dimensional complex force field composed of centrifugal and orthogonal gravitational force, and the third employing the apparatus placed at any orientation including horizontal and upright CCC apparatus at high revolutional speed above 300rpm in the centrifugal force field. Therefore, we should consider the effect of gravitation force on CCC at the selection of proper operational conditions according to properties of two immiscible phases, the desired revolutional speed and the parameters of apparatus in order to utilize efficiently the effect of gravitational force. These analyses and results will benefit to improve the efficiency of CCC separation, especially large-scale industrial preparation.     

应用立式逆流色谱和反相液相色谱制备性分离荜菝中的酰胺类化合物

A versatile countercurrent chromatography with upright type-J multilayer coil planet centrifuge,named upright countercurrent chromatography (UCCC),was applied to the isolation and purification of amides from Piper Longum L., which is widely used as an anodyne and a treatment for stomach disease in China. After saponification by KOH of the ethanol extracts solution of crud drug "Piper Longi Fructus", the fruits of Piper Longum L., the remaining solution was extracted with petroleum ether and red crude oil was obtained. Using 2.5 g red crude oil as sample, the preparative UCCC with a two-phase system composed of petroleum ether (b. p. 60 ～ 90 ℃)-ethyl acetate-tetrachloromethanemethanol-water ( 1: 1: 8: 6: 1, v/v) was successfully performed, yielding ten fractions. Then these fractions were further purified by use of reversed-phase liquid chromatography with a glass column of 500 × 10 mm I. D.packed with reversed - phase silica gel. As a result,nine amides with more than 98% purity, i.e., (2E,4E)-N-isobutyl-eicosa-2,4-dienamide, (2E, 4E, 8Z)-N-isobutyl-eicosa-2,4,8-trienamide, (2E, 4E, 8Z)-N-isobutyl-ocatadeca-2,4,8-trienamide, guineensine, pipemonaline, ( 2E, 4E )-N-isobutyl-2, 4-didecadienamide, piperine, piperanine, and piperlonguminine were isolated preparatively.     

Preparative isolation and purification of amides from the fruits of Piper longum L. by upright counter-current chromatography and reversed-phase liquid chromatography

A versatile counter-current chromatography (CCC) with upright type-J multilayer coil planet centrifuge, named upright CCC, was applied to the isolation and purification of amides from Piper longum L., which is widely used as an anodyne and a treatment for stomach disease in China. After the saponification by KOH of the ethanol extracts solution of 15 kg of crude drug "Piper Longi Fructus", the fruits of P. longum L., the solution was extracted with light petroleum and 500 g of red crude oil was obtained. Using 2.5 g of red crude oil as sample, the preparative upright CCC with a two-phase system composed of light petroleum (bp 60-90 degrees C)-ethyl acetate-tetrachloromethane-methanol-water (1:1:8:6:1, v/v) was successfully performed, which yielded nine fractions. Then these fractions were further purified by use of reversed-phase liquid chromatography (RPLC) with a glass column of 500 x 10 mm i.d. packed with reversed-phase silica gel. As a result, nine target amides with over 95% purity, i.e., 50 mg of (2E,4E)-N-isobutyl-eicosa-2,4-dienamide, 150 mg of (2E,4E,14Z)-N-isobutyl-eicosa-2,4,14-trienamide, 110 mg of (2E,4E,12Z)-N-isobutyl-ocatadeca-2,4,12-trienamide, 50 mg of guineensine, 60 mg of pipernonaline, 75 mg of pellitorine, 63 mg of piperine, 45 mg of piperanine, and 40 mg of piperlonguminine were isolated, respectively. Structures of all compounds were identified by electrospray ionization MS, electron impact ionization MS, one- and two-dimensional NMR spectra.     

Preparative isolation and purification of celastrol from Celastrus orbiculatus Thunb. by a new counter-current chromatography method with an upright coil planet centrifuge

A new counter-current chromatography (CCC) method with an upright coil planet centrifuge, which holds four identical multilayer coil columns in the symmetrical positions around the centrifuge axis, was applied to the isolation and purification of celastrol from the roots of Celastrus orbiculatus Thunb. The crude celastrol was obtained by elution with light petroleum from ethanol extracts using 15 cm x 5 cm i.d. silica gel flash chromatography. Preparative CCC with a two-phase system composed of light petroleum (bp 60-90 degrees C)-ethyl acetate-tetrachloromethane-methanol-water (1:1:8:6:1, v/v) was successfully performed, yielding 798 mg celastrol at 99.5% purity from 1020 mg of the crude sample in one step separation.     

Countercurrent Chromatography Using a Toroidal Coil Planet-Centrifuge: A Comparative Study of the Separation of Organelles Using Aqueous Two-Phase Partition

Abstract

A toroidal coil planet centrifuge is described and compared with other countercurrent chromatography (CCC) and countercurrent distribution (CCD) techniques. The basis of separation is partition in aqueous two-phase polymer systems, with each method assessed by fractionating rat liver organelles. The size and ease of operation of the toroidal coil planet centrifuge gave significant advantages over conventional CCD systems achieving equivalent resolution in a fraction of the time.     

Evaluation of Analytical Countercurrent Chromatographs: High-Speed Countercurrent Chromatograph-4000 Vs. Analytical Toroidal Coil Centrifuge

Abstract

Performance of two countercurrent chromatographic models, high speed countercurrent chromatograph (HSCCC-4000) and analytical toroidal coil centrifuge (TCC), is evaluated in terms of theoretical plate number, resolution factor and separation time to assess their analytical capability. A series of experiments was conducted to investigate the effects of internal diameter and length of the coiled column, and flow rate of the mobile phase on the separation of indole auxins in two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at different volume ratios. The three components of indole auxins were completely resolved in 16 min with a HSCCC system equipped with a multilayer coil of a 0.55 mm I.D. PTFE tube with theoretical plates ranging from 1290 to 829. Similar separation was achieved in 24 min with a TCC systems equipped with a 0.3 mm I.D. PTFE tube with theoretical plates ranging from 1811 to 969. It is concluded that both systems have comparable analytical capability at the present stage of development.     

Using the liquid nature of the stationary phase in countercurrent chromatography. IV. The cocurrent CCC method

The retention volumes of solutes in countercurrent chromatography (CCC) are directly proportional to their distribution coefficients, K(D) in the biphasic liquid system used as mobile and stationary phase in the CCC column. The cocurrent CCC method consists in putting the liquid "stationary" phase in slow motion in the same direction as the mobile phase. A mixture of five steroid compounds of widely differing polarities was used as a test mixture to evaluate the capabilities of the method with the biphasic liquid system made of water/methanol/ethyl acetate/heptane 6/5/6/5 (v/v) and a 53 mL CCC column of the coil planet centrifuge type. It is shown that the chromatographic resolution obtained in cocurrent CCC is very good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. Pushing the method at its limits, it is demonstrated that the five steroids can still be (partly) separated when the flow rate of the two liquid phases is the same (2 mL/min). This is due to the higher volume of upper phase (72% of the column volume) contained inside the CCC column producing a lower linear speed compared to the aqueous lower phase linear speed. The capabilities of the cocurrent CCC method compare well with those of the gradient elution method in HPLC. Continuous detection is a problem due to the fact that two immiscible liquid phases elute from the column. It was partly solved using an evaporative light scattering detector.     

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 increased 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, V and X derived from Clostridium hystolyticum were separated from other proteins and colored small molecular weight compounds present in the crude commercial sample, while collagenase N-2 and S-1 from Streptomyces parvulus subsp. citrinus were eluted with impurities at the solvent front with the upper phase. The collagenase from C. hystolyticum 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.     

应用新型立式逆流色谱制备分离南蛇藤中的南蛇藤素

A versatile countercurrent chromatography with upright multilayer coil planet centrifuge, named upright countercurrent chromatography (UCCC), was applied to the isolation and purification of celastrol from the roots of Celastrus orbiculatus Thunb. The crude celastrol was obtained by elution with petroleum ether from ethanol extracts using a 15 cm length and 5 cm I.D. of silica gel flash chromatography. Preparative UCCC (Fig. 1) with a two-phase system composed of petroleum ether (b. p. 60 ～ 90 ℃ )-ethyl acetate-tetrachloromethanemethanol-water ( 1:1:8:6: 1, v/v) was successfully performed, yielding 705 mg celastrol at 99.5 % purity from 1020 rng of the crude extract in one step separation.