Isolation of sutherlandins A, B, C and D from Sutherlandia frutescens (L.) R. Br. by counter-current chromatography using spiral tubing support rotors.

Spiral countercurrent-chromatography has great potential for improving the capacity and efficiency of purification of secondary metabolites, and here we describe applications useful for the isolation of flavonoids from the widely used South African medicinal plant, Sutherlandia frutescens (L.) R. Br. In the spiral tubing support rotor, STS-4 for high-speed counter-current chromatography, several polar butanol aqueous solvent systems were selected using a logK plot, and the novel flavonol glycosides (sutherlandins A-D) were well separated by the optimized solvent system (ethyl acetate:n-butanol:acetic acid:water; 5:1:0.3:6 by vol.). The yield of purified flavonoids from 0.9g extract varied from 8.6mg to 54mg of the sutherlandins for a total of 85.3mg. The same extract was fractionated in the new STS-12 rotor of the same outside dimensions but with more radial channels forming 12 loops of the tubing instead of 4. The rotor holds more layers and increased length of tubing. From 0.9g extract the STS-12 rotor yielded more recovery of 110.4mg total with amounts varying from 11.2mg to 64mg of the sutherlandins and apparent increased separation efficiency as noted by less volume of each fraction peak. Thus from 1-g amounts of extract, good recovery of the flavonoids was achieved in the butanol aqueous solvent system.

Spiral countercurrent chromatography.

For many years, high-speed countercurrent chromatography conducted in open tubing coils has been widely used for the separation of natural and synthetic compounds. In this method, the retention of the stationary phase is solely provided by the Archimedean screw effect by rotating the coiled column in the centrifugal force field. However, the system fails to retain enough of the stationary phase for polar solvent systems such as the aqueous-aqueous polymer phase systems. To address this problem, the geometry of the coiled channel was modified to a spiral configuration so that the system could utilize the radially acting centrifugal force. This successfully improved the retention of the stationary phase. Two different types of spiral columns were fabricated: the spiral disk assembly, made by stacking multiple plastic disks with single or four interwoven spiral channels connected in series, and the spiral tube assembly, made by inserting the tetrafluoroethylene tubing into a spiral frame (spiral tube support). The capabilities of these column assemblies were successfully demonstrated by separations of peptides and proteins with polar two-phase solvent systems whose stationary phases had not been well retained in the earlier multilayer coil separation column for high-speed countercurrent chromatography.

Application of centrifugal precipitation chromatography and high-speed counter-current chromatography equipped with a spiral tubing support rotor for the isolation and partial characterization of carotenoid cleavage-like enzymes in Enteromorpha compressa (L.) Nees.

Centrifugal precipitation chromatography and a high-speed counter-current chromatography system equipped with a spiral tubing support rotor (spHSCCC) were successfully applied for the identification and isolation of carotenoid cleavage-like enzymes from Enteromorpha compressa (L.) Nees. This is the first study separating active enzymes from a complex natural matrix by spHSCCC. The target enzymes were identified after fractionation of the proteins in an acetone Tris-buffer gradient by centrifugal precipitation chromatography. Also, an aqueous two-phase solvent system consisting of PEG 1000 and mono- and dibasic potassium phosphate was used for the isolation of the enzymes by spHSCCC. The purified fractions contained two proteins of 65 and 72 kDa, respectively. The enzymes could cleave ß-carotene and ß-apo-8'-carotenal to produce ß-ionone.

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.

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.

Vortex counter-current chromatography.

A novel counter-current chromatographic system is developed by mounting a vortex column on a type-I coil planet centrifuge. The column is fabricated from a high-density polyethylene disk (16 cm diameter and 5 cm thick) by making multiple holes of various diameters (3-12.5 mm) each arranged in a circle and connected with narrow transfer ducts. The performance of this vortex column is tested with three different two-phase solvent systems with a broad range in hydrophobicity. The results indicated that the smallest diameter column (3mm diameter, 120 units with 42.8 ml capacity) yielded the best separation with the height equivalent to a theoretical plate of 2 cm compared with 20 cm required by the conventional multilayer coil column of high-speed CCC. By avoiding the use of an Archimedean Screw Force, the system shows a low column pressure which would permit safe operation of a large preparative column without a risk of leakage of solvent and column damage.