Spiral Counter-current Chromatography Research Articles

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 Enrichment, Characterization, and Assays of Carbon Nanotube Chiralities for Use in Biosensors.

Single-walled carbon nanotubes (SWCNTs) are synthetic materials that hold great promise for electronics that are smaller and more versatile than the current silica-based technologies. But as-produced SWCNTs are generally a mixture of nanotubes with different structures that have vastly different properties. Separating these SWCNTs from multiwalled and metallic carbon nanotubes is vital to explore their individual properties and commercial utility ranging from optics to semiconductors. Compounding the problem of commercial investigation is that the semiconducting SWCNTs are also a mixture of different diameters and/or chiralities with different properties. Analyzing properties of enriched semiconducting SWCNT chiralities has only recently been possible through separation techniques such as aqueous two-phase solvent systems. Our study illustrates a semipreparative spiral countercurrent chromatography (CCC) separation of a commercial mixture of SWCNTs into distinct enriched fractions. A new mixer–settler spiral disk rotor was applied in this study, in which we compare the enriched SWCNTs for their effectiveness in biosensors with a high-throughput model assay, followed by antibody-mediated detection of Escherichia coli. Our results demonstrate that CCC-enriched responsive SWCNTs for biosensors can be used in our model assay, as well as for the detection of E. coli. To date, we believe that this is the first study along with Liu et al. [Chirality-controlled synthesis of single-wall carbon nanotubes using vapour-phase epitaxy. Nat. Commun.2012, 3, 119923149724] to demonstrate a specific utility of separated SWCNT species.

Purification of semiconducting single-walled carbon nanotubes by spiral counter-current chromatography

Over the last decade man-made carbon nanostructures have shown great promise in electronic applications, but they are produced as very heterogeneous mixtures with different properties so the achievement of a significant commercial application has been elusive. The dimensions of single-wall carbon nanotubes are generally a nanometer wide, up to hundreds of microns long and the carbon nanotubes have anisotropic structures. They are processed to have shorter lengths but they need to be sorted by diameter and chirality. Thus counter-current chromatography methods developed for large molecules are applied to separate these compounds. A modified mixer-settler spiral CCC rotor made with 3 D printed disks was used with a polyethylene glycol-dextran 2-phase solvent system and a surfactant gradient to purify the major species in a commercial preparation. We isolated the semi-conducting single walled carbon nanotube chiral species identified by UV spectral analysis. The further development of spiral counter-current chromatography instrumentation and methods will enable the scalable purification of carbon nanotubes useful for the next generation electronics.

Spiral counter-current chromatography: Design, development, application, and challenges.

Depending on the rapid growth in the radial gradient of the centrifugal force, spiral counter-current chromatography can greatly improve the retention of stationary phase, especially for the aqueous two-phase systems with ultra-polar and high viscosity that are not well retained in the conventional multilayer coils counter-current chromatography. As a result, it is an attractive and alternative technology that is suited for separation of hydrophilic compounds and has led to many exciting progress in recent years. This review presents the recent advances and applications of spiral counter-current chromatography, including its major benefits and limitations, some novel methods to improve the separation efficiency and its applications in separation of real samples. In addition, the remaining challenges and future perspectives on development of spiral counter-current chromatography also are proposed in this article.

Isolation of the Predominant Cycloartane Glycoside, Sutherlandioside B, from Sutherlandia frutescens (L.) R.Br. by Spiral Countercurrent Chromatography

The introduction to high-speed countercurrent chromatography of a spiral tubing support rotor has enabled the application of more polar volatile solvent systems for natural products separation, especially water soluble compounds and their metabolites. Here is reported the use of spiral countercurrent chromatography with the spiral tubing support rotor to fractionate extracts of the African medicinal plant Sutherlandia frutescens (L.) R.Br. A two-phase solvent system of ethyl acetate, methanol, water, and n-butanol, with the lower aqueous phase mobile, separated sutherlandioside B efficiently from other compounds. The purity of sutherlandioside B was determined by high-performance liquid chromatography and mass spectrometry analysis. Sufficient quantities of sutherlandioside B can be purified by this method for use in cell culture assays and the development of a sensitive antibody by which to detect the consumption and metabolism in vivo of this novel botanical.

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.

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.

Preparative Separation of Picralima Alkaloids using a New Flat-Twisted Tubing in pH-Zone-Refining Countercurrent Chromatography Mode

A novel column for spiral countercurrent chromatography (CCC) has been designed to interrupt the laminar flow of the mobile phase through the tubing and continuously mix the two phases throughout the column [1]. Flat-twisted tubing system was originally introduced for purification of large molecules (proteins and peptides). Its application in the separation of small molecules is presented for the first time. Here pH-zone-refining CCC is successfully applied to the preparative separation of Picralima alkaloids using this new tubing filled in the spiral tubing support rotor (STS, CC Biotech, Rockville MD). The seeds of Picralima nitida (Apocynaceae), popularly known in African ethnomedicine for its antimalarial activity, were extracted successively with solvents of increasing polarity to obtain hexane, dichloromethane (DCM) and methanol (MeOH) extracts. The experiment was performed with a two-phase solvent system composed of t-methyl-butyl ether-n-butanol-acetonitrile-water at an optimized volume ratio of 2:2:1:5 (v/v) where triethylamine (5mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. Two separation runs of 4.0g each of the DCM and MeOH extracts yielded six major indole alkaloids including akuammine, akuammicine, alstonine, picraline, Melinonine A and akuammidine with purities ranging from 90% (Melinonine A) to 98% (akuammine) as determined by HPLC. Overall, the results revealed that the new Flat-twisted tubing system yielded high partition efficiency with a satisfactory level of stationary phase retention in a short elution time.

Separation of Peptides by Spiral Countercurrent Chromatography

Spiral countercurrent chromatography (CCC) utilizes a new separation rotor composed of 8 high density polyethylene plates with spiral flow channels, mounted in the type‐J coil planet centrifuge (CPC). Synthetic peptides were evaluated for partition coefficients in various solvent systems. Approximately 30 mg amounts were chromatographed and the fractions analyzed for peptide content and purity. The solvent systems which contained sec‐butanol or n‐butanol and the mobile phase selected produced good purification. The stationary phase was highly retained over 60%, thus the spiral separation coil is able to retain these more viscous, low interfacial tension solvents that have not been able to be used well in the multi‐layer coil for high speed CCC.

Numerical model for the investigation of dual-flow in a spiral counter-current chromatography column

In dual-flow counter-current chromatography (DF-CCC), the two immiscible liquids are flowing in opposite directions in the coil. The method allows for the continuous separation of two solutes. In this study a numerical model was developed to allow for the detailed investigation of flow in such columns. The mesh model of the presented DF spiral column was developed in line with an existing experimental model. The paper presents results during the early filling stages for different rotation directions. These clearly illustrate the performance of the developed model by (1) confirming the importance of flowing the lighter phase from tail to head and the heavier phase from head to tail and (2) by visualising mixing waves and the recognised back and forth “swish-swash” motion as present in CCC in that operating mode.