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.     

Toroidal Coil Countercurrent Chromatography: A Fast Simple Alternative to Countercurrent Distribution Using Aqueous Two Phase Partition: Principles,Theory, and Apparatus

Abstract

The principles, theoretical basis and equipment for continuous two phase toroidal coil chromatography are described. Rat liver homogenates were subjected to analytical subcellular fractionation by toroidal coil chromatography in a phase mixture of 3.3% (w/w) dextran T500, 5.4% (w/w) poly(ethylene glycol) 6000, 10 mM sodium phosphate-phosphoric acid buffer, pH 7.4, in 0.26 M sucrose containing 0.05 mM Na₂EDTA and 1 mM ethanol. The distribution of organelles, as reflected by their marker enzymes, was compared to that obtained by discrete counter-current partition in a 17 transfer apparatus. Toroidal coil chromatography showed enhanced resolution of certain organelles. In particular, almost complete separation of plasma membrane from endoplasmic reticulum was achieved and some resolution of plasma membrane from lysosomes was obtained. It is concluded that toroidal coil chromatography offers a potentially useful alternative approach to organelle separation techniques.     

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.     

Plant Hormone Analysis by Countercurrent Chromatography

Abstract

Countercurrent chromatography (CCC) has been successfully applied for the separation of plant hormones; namely, indole auxins, gibberellins, cytokinins and abscisic acid. In our present study three different types of CCC devices were evaluated for their performance in separation of plant hormones with a special emphasis on analysis and purification of abscisic acid (ABA). A large-scale preparative CCC apparatus consisting of a slowly rotating coil assembly was used for preliminary separations of ABA from a large volume of crude plant extracts. The toroidal coil planet centrifuge (CPC) for analytical-scale separations was subsequently applied for purification of ABA, the final confirmation being obtained by HPLC and combined gas chromatographic-mass spectrometric method. This two-step procedure utilizing preparative CCC and toroidal CPC was successfully applied for determination of ABA content in several plant tissues. A recently introduced high-speed CCC apparatus was tested for semipreparative separation of ABA and indole-3-acetic acid. The method yielded high peak resolution within 2 hours.     

Cell Separations on the Countercurrent Chromatograph

Abstract

Separation of cells differing only subtly has been achieved by partitioning between the two phases formed by solution of dextran and polyethylene glycol in water. Cell populations which have related, but not identical, surface properties seldom exhibit sufficiently different partition behavior to be separated in a single extraction. In such cases, repeated partitions are carried out via countercurrent distribution or countercurrent chromatography to effect the separation. Potential advantages of countercurrent chromatography are its ease and rapidity of operation. In the present work we describe our approach to determining and possibly improving the efficiency of polymer phase partitioning by countercurrent chromatography.     

Purification of Solid-Phase Synthesized Peptides on the Coil Planet Centrifuge

Abstract

The analytical flow-through coil planet centrifuge, an instrument for countercurrent chromatography, performs the preparative purification of synthetic peptides. Various two-phase solvent systems have been tried with either phase mobile to purify many synthesized peptides. A series of N-terminal fragment peptides of cholecystokinin octapeptide (CCK 26–33) were synthesized by solid-phase techniques and purified on the coil planet centrifuge. The peptides were sulfated and chromatographed again. For hydrophobic peptides, purification is effected in solvent systems with a mobile aqueous phase. The n-butanol, acetic acid and water system (4:1:5 by volume) with the lower phase mobile was utilized. For sulfated peptides, the neutral system, 0.2 M ammonium acetate and n-butanol was generally applied.     

Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems

Dual high-speed countercurrent chromatography (dual CCC) literally permits countercurrent flow of two immiscible solvent phases continuously through the coiled column for separation of solutes according to their partition coefficients. Application of this technique has been successfully demonstrated by separation of analytes by gas–liquid and liquid–liquid two-phase systems. However, the method cannot be directly applied to the system with a set of coiled columns connected in series, since the countercurrent process is interrupted at the junction between the columns. However, this problem can be solved by intermittent dual CCC by eluting each phase alternately through the opposite ends of the separation column. This mode of application has an advantage over the conventional dual CCC in that the method can be applied to all types of CCC systems including hydrostatic equilibrium systems such as toroidal coil CCC and centrifugal partition chromatography. Recently, the application of this method to high-speed CCC (hydrodynamic system) has been demonstrated for separation of natural products by Hewitson et al. using a set of conventional multilayer coil separation columns connected in series. Here, we have developed a mathematical model for this intermittent dual CCC system to predict retention time of the analytes, and using a simplified model system the validity of the model is justified by a series of basic studies on both hydrodynamic and hydrostatic CCC systems with a computer-programmed single sliding valve. The present method has been successfully applied to spiral tube assembly high-speed CCC (hydrodynamic system) and toroidal coil CCC (hydrostatic system) for separation of DNP-amino acid samples with two biphasic solvent systems composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1 and 4:5:4:5, v/v).     

Foam Countercurrent Chromatography Based on Dual Counter-Current System

Abstract

A novel method of performing foam separation is described. The method utilizes a gas-liquid dual countercurrent flow through a helical column subjected to a particular type of synchronous planetary motion. Samples introduced at the middle portion of the column, in either batch or continuous mode, are separated according to the foam affinity. Any material having an affinity to the foam is quickly carried with the foaming stream and eluted through one end of the column whereas other materials are carried with the liquid stream in the opposite direction and eluted out through the other end of the column. Capability of this foam countercurrent chromatographic method is demonstrated on separations of rhodamine B and Evans blue with an anionic surfactant, SDS, as a collector of rhodamine B. Successful preliminary separation of protein samples, BSA and sheep hemoglobin, indicates that the present method may be effectively applied to separation and purification of various biological samples such as enzymes, membrane receptors, etc.     

Synergistic Use of Countercurrent Chromatography and High Performance Liquid Chromatography for the Purification of Synthetic Peptides

Abstract

Examples are given demonstrating how countercurrent chromatography (CCC) and high performance liquid chromatography (HPLC) can be used together to purify synthetic peptides. In one example, CCC provided a preliminary purification of Met-Arg-Asp-Val-Val-Leu-Phe-Glu-Lys by enabling separation of ultraviolet absorbing, ninhydrin-negative material from the desired peptide. Final purification was achieved with HPLC without risk of harming the HPLC column. In a second example Tyr-Ala-Ala-Nle-Ala-Ala was completely purified by CCC with the CCC separation rapidly and conveniently monitored by HPLC. CCC appears to be a very useful technique for the peptide chemist.     

Countercurrent Chromatography for Isolation of Flavonol Glycosides From Ginkgo Biloba Leaves

Abstract

A procedure which combined countercurrent chromatography with gradient elution and preparative high-performance liquid chromatography was developed for the isolation and the purification of the seven predominant flavonol glycosides from Ginkgo biloba leaves.     

Modelling Affinity Chromatography

Abstract

Affinity chromatography plays a significant role in the separation and purification of biologically active macromolecules in laboratory and large-scale applications. There is a need for models which could be used to predict accurately the dynamic behavior of affinity chromatography separations, in order to permit the design, optimization, control, and process scale-up of affinity chromatography systems. Furthermore, the construction and use of such models will contribute to a better fundamental understanding of the physicochemical and biospecific mechanisms involved in affinity chromatography processes. The parameters of the models should be obtainable by using information from a small number of experiments.

This work reviews the modeling of affinity chromatography, and presents general models that could be used to describe the dynamic behavior of the adsorption, wash, and elution stages of affinity chromatography systems. Certain model structures, modeling approaches and operational strategies for systems having porous or nonporous adsorbent particles are also suggested, and experiments are proposed whose data are necessary for parameter estimation and model discrimination studies in affinity chromatography.

Particular emphasis is given to :he modeling of the intrinsic mechanisms of intraparticle diffusion, adsorption, and desorption, because the intrinsic mechanisms are normally independent of the mode of operation (i.e., batch, fixed bed, fluidized bed, continuous countercurrent, or others).     

Optimization of Solvent Mixture Compositions for High Speed Countercurrent Distribution

Abstract

Seven solvents considered appropriate for use with relatively sensitive biosynthetic products undergoing separation by multilayer coil planet centrifuge high speed countercurrent distribution (HSCCD) have been evaluated in a new and systematic procedure for optimal solvent selection. the new method was illustrated by a complete HSCCD separation of five closely related dipeptides.     

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.     

HPLC Purification of Solid-Phase Generated Synthetic Bombesin

Abstract

A scheme based on ion-exchange and reverse-phase high pressure liquid chromatography has been utilized for the semi-preparative and preparative purification of the solid phase generated model peptide bombesin. The final product showed a purity ≥ 99% in analytical reverse-phase high pressure liquid chromatography and was identical to authentic bombesin as demonstrated by different physico-chemical and biological criteria. The results are discussed and compared to those obtained using countercurrent chromatography.     

Efficient separation of tocopherol homologues in vegetable oil by ionic‐liquid‐based countercurrent chromatography using a non‐aqueous biphasic system

Tocopherol homologues are important fat‐soluble bioactive compounds with high nutritional value. However, it is of great challenge to separate these homologues because of their high structural similarities. In this work, ionic‐liquid‐based countercurrent chromatography was used for the separation and purification of tocopherol homologues. Conventional countercurrent chromatography and ionic‐liquid‐based countercurrent chromatography solvent systems were evaluated in respect of partition coefficient, separation factor, and stationary phase retention factor to separate these targets. Kind of ionic liquids, amount of ionic liquid, and sample amount were systematically optimized. A novel countercurrent chromatography non‐aqueous biphasic system composed of n‐hexane‐methanol‐1‐butyl‐3‐methylimidazolium chloride was established. The baseline separation of tocopherol mixtures was obtained in one cycle process. The ionic liquid played a key role in the countercurrent chromatography separation, which resulted in difference of partition behavior of individual tocopherol in the whole system through different hydrogen‐bonding affinity. Finally, n‐hexane‐methanol‐1‐butyl‐3‐methylimidazolium chloride (5:5:3, v/v) water‐free biphasic system was successfully applied to separate tocopherol homologues from vegetable oil that was not achieved beforehand. This method can be widely employed to separate many similar molecules such as tocotrienols, tocomonoenols, and marine‐derived tocopherol in food samples.     

Quick method for separating target compounds from the bark of Maqian (Zanthoxylum myriacanthum var. pubescens) by high‐performance countercurrent chromatography

Choosing a suitable solvent system for a countercurrent chromatography separation presents a challenge for many researchers. In this study, we introduce a quick method of separating a target compound from the bark of Zanthoxylum myriacanthum var. pubescens by countercurrent chromatography. This method relies on the thin‐layer chromatography based generally useful estimation of solvent systems. This paper will present how to quickly choose a suitable solvent system with a thin‐layer chromatography based generally useful estimation of solvent systems working chart. O‐Methyltembamide ( 1 ) was enriched by countercurrent chromatography using n‐hexane/ethyl acetate/methanol/water (6:4:6:4) as the solvent system. Further purification was achieved by high‐performance liquid chromatography with purities of 98.2% from Z. myriacanthum var. pubescens bark.     

Preparative isolation and purification of hainanmurpanin,meranzin, and phebalosin from leaves of Murraya exotica L. using supercritical fluid extraction combined with consecutive high‐speed countercurrent chromatography

The objective of this study was to develop a consecutive preparation method for the isolation and purification of hainanmurpanin, meranzin, and phebalosin from leaves of Murraya exotica L. The process involved supercritical fluid extraction with CO₂, solvent extraction, and two‐step high‐speed countercurrent chromatography. Pressure, temperature, and the volume of entrainer were optimized as 27 MPa, 52°C, and 60 mL by response surface methodology in supercritical fluid extraction with CO₂, and the yield of the crude extracts was 7.91 g from 100 g of leaves. Subsequently, 80% methanol/water was used to extract and condense the three compounds from the crude extracts, and 4.23 g of methanol/water extracts was obtained. Then, a two‐step high‐speed countercurrent chromatography procedure was developed for the isolation of the three target compounds from methanol/water extracts, including conventional high‐speed countercurrent chromatography for further enrichment and consecutive high‐speed countercurrent chromatography for purification. The yield of concentrates from high‐speed countercurrent chromatography was 2.50 g from 4.23 g of methanol/water extracts. Finally, the consecutive high‐speed countercurrent chromatography produced 103.2 mg of hainanmurpanin, 244.7 mg of meranzin, and 255.4 mg of phebalosin with purities up to 97.66, 99.36, and 98.64%, respectively, from 900 mg of high‐speed countercurrent chromatography concentrates in one run of three consecutive sample loadings without exchanging a solvent system.     

Chromatography of AC-ASP-TYR-MET-GLY-TRP-MET-ASP-NH2 on the Horizontal flow-Through Coil Planet Centrifuge and the High-Speed Multi-Layer Coil Planet Centrifuge

Abstract

The peptide Ac-Asp-Tyr-Met-Gly-Trp-Met-Asp-NH₂ was purified by countercurrent chromatography in the horizontal flow-through coil planet centrifuge. The solvent system used was ammonium acetate, pH 8.5 and n-butanol (1:1 by volume). The high pH served to maintain the peptide in solution. When the upper phase was utilized as the mobile phase better separation of the peptide from impurities resulted. The peptide was also chromatographed in a new apparatus, the high-speed multi-layer coil planet centrifuge. With the lower phase mobile and at a higher temperature, the peptide was fractionated very rapidly in 30 min compared to 7 hr on the other instrument.     

Online hyphenation of extraction,Sephadex LH‐20 column chromatography,and high‐speed countercurrent chromatography: A highly efficient strategy for the preparative separation of andrographolide from Andrographis paniculata in a single step

A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH‐20 column chromatography combined with high‐speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH‐20 column directly to cut the nontarget fractions followed by the second‐dimensional high‐speed countercurrent chromatography, hyphenated by a six‐port valve equipped at the post‐end of Sephadex LH‐20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant.     

Application of High Speed Countercurrent Chromatography/Thermospray Mass Spectrometry for the Analysis of Bio-Active Triterpenoic Acids from Boswellia Carterh

Abstract

The versatility and high resolving power of countercurrent chromatography has been demonstrated with a newly developed analytical high speed planet centrifuge system (HSCCC). Interfacing countercurrent chromatography with mass spectrometry (MS) provides a new analytical methodology which Integrates the advantages of countercurrent chromatography with the low detection limit and identification capability of mass spectrometry. The capability of thermo-spray HSCCC/MS has been successfully demonstrated in the identification of plant alkaloids and lignans. In this paper, the technigue proved useful in identifying and validating the bio-active and structural closely related triterpenoid carboxylic acids from a crude extract of Boswellia carterii (Burseraceae). Thermospray HSCCC/MS can become a useful and complementary method to thermospray HPLC/MS for the analysis of nonvolatile or thermally unstable molecules.