Closed-Loop Recycling Dual-Mode Counter-Current Chromatography with Specified Sample Loading Durations: Modeling of Preparative and Industrial-Scale Separations

We previously reported on a new counter-current chromatography (CCC) operating mode called closed-loop recycling dual-mode counter-current chromatography (CLR DM CCC), which incorporates the advantages of closed-loop recycling (CLR) and dual-mode (DM) counter-current chromatography and includes sequential separation of compounds in the closed-loop recycling mode with the mobile x-phase and in the inverted-phase counter-current mode with the mobile y-phase. The theoretical analysis of several implementations of this separation method was carried out under impulse sample injection conditions. This study is dedicated to the further development of CLR DM CCC theory applied to preparative and industrial separations, where high-throughput operation is required. Large sample volumes can be loaded via continuous loading within a specified time. To simulate CLR DM CCC separations with specified sample loading durations, equations are developed and presented in “Mathcad” software.     

Universal counter-current chromatography modelling based on counter-current distribution

There is clearly a need for a model which is versatile enough to take into account the numerous operating modes and pump out procedures that can be used with counter-current chromatography (CCC). This paper will describe a universal model for counter-current chromatography based on counter-current distribution. The model is validated with real separations from the literature and against established CCC partition theory. This universal model is proven to give good results for isocratic flow modes, as well as for co-current CCC and dual flow CCC, and will likely also give good results for other modes such as intermittent CCC.     

Recent advances in counter-current chromatography

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.     

Purification of drugs from biological fluids by counter-current chromatography

Experiments were performed to demonstrate the potential of counter-current chromatography (CCC) for the isolation of drugs and their metabolites from biological matrices relevant to the metabolism studies of pharmaceutical research. Examples of typical drugs are spiked into biological media ex vivo to provide test samples for analysis. A mass spectrometer hyphenated to a CCC allows for the detection of small molecule drugs within the matrix through selected ion monitoring, and fraction collection can provide material for further structural elucidation by NMR.     

Modelling counter-current and dual counter-current chromatography using longitudinal mixing cell and eluting counter-current distribution models

Two well known approaches are considered to analyze the processes of counter-current and dual counter-current chromatography: the longitudinal mixing cell model and the Craig's counter-current distribution model. The cell model represents perfectly mixed, equally sized cells in series. The number of cells characterizes the rates of longitudinal mixing in the stationary and mobile phases. In the eluting counter-current distribution (CCD) model, the CCC process is considered as a continuous form of Craig's counter-current distribution. For a cascade of equilibrium stages theoretical elution profiles of the CCC process by using the CCD and cell model approaches have been compared. It is shown that in general, distribution functions of the CCD and cell models differ. It is established that the distribution of a solute between two solvent phases in the dual CCC process is determined by the extraction factor c, the total number of equilibrium stages n and the position of the sample inlet m by the equation Q(x)=(1-c(m))/(1-c(n+1)) with c=F(2)K(D)/F(1) (K(D), F(1), F(2) and Q(x) are the distribution constant, the phase flow-rates and the portion of solute eluted by the first phase, respectively).     

逆流色谱仪器装置的研究进展

逆流色谱是一种快速、高效、无固定相载体的新型液-液分配色谱技术.综述了高速逆流色谱、双向逆流色谱、正交轴逆流色谱、pH-区带精制逆流色谱及螺线型圆盘柱式逆流色谱仪器装置的研究进展,介绍了高速逆流色谱-质谱联用技术、高速逆流色谱.蒸发光散射联用技术、高速逆流色谱.高效液相色谱联用技术及二维逆流色谱技术的发展状况.     

Separation of alkaloids from herbs using high-speed counter-current chromatography

Alkaloids represent a most widespread group of bioactive natural products. Because of their alkalinity and structural diversity, the fractionation and purification of the alkaloids from herbs can often present a number of practical difficulties using the conventional chromatographic techniques. High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatography with a support-free liquid stationary phase, and is gaining more and more popularity as a viable separation technique for bioactive compounds from natural resources. In the present review, focus is placed on the separation of alkaloids by both conventional HSCCC and pH-zone-refining counter-current chromatography (CCC) techniques from herbs. The review presents the separation of over 120 different alkaloid compounds from more than 30 plant species by the conventional HSCCC and pH-zone-refining CCC. Based on the data from the literature, the proper solvent systems for the separation of alkaloids by the conventional HSCCC and pH-zone-refining CCC are also summarized.     

Continuous counter-current extraction on an industrial sample using dual-flow counter-current chromatography

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.     

Counter-current chromatography based analysis of synergy in an anti-tuberculosis ethnobotanical

The crude extract of an Alaskan ethnobotanical plant, Oplopanax horridus, was subjected to counter-current chromatography (CCC), and the selected active regions were evaluated for their synergistic effects with an in vitro model of anti-tubercular efficacy. CCC as a support-free high-resolution separation method was employed to preclude potential irreversible absorption to a solid stationary phase. The microplate Alamar blue assay and the isobole method were used to measure the biological activity and eliminate dose-response dependent errors, respectively. Using the combination of CCC, bioassay and isobole method, significant synergistic effects were observed. Among the entire polarity range, fractions with distribution constant between 0.44 and 0.81 showed the most synergistic enhancement with an increase in potency by 108% for the recombined fractions.     

Recent progress on the industrial scale-up of counter-current chromatography

The pharmaceutical industries are looking for rapid methods of purification and predictable scale-up for their drug development process that will cut their costs and enable them to reduce the time to market. In this paper, recent progress is reviewed in the development and demonstration of two types of industrial scale centrifugal liquid-liquid chromatography: hydrostatic and hydrodynamic. Industrial scale hydrostatic processes by Partus Technologies and Armen Instrument are just emerging. Results demonstrating scalability are presented for hydrodynamic processes by Dynamic Extractions. The review concludes that the time is now right, with this appropriate commercial support, for high performance counter-current chromatography to emerge as a major enabling technology for industry.     

Model for spiral columns and stationary phase retention in synchronous coil planet centrifuges

Stationary phase retention in a synchronous coil planet centrifuge or high-speed counter-current chromatography (CCC) relies on the interplay of hydrostatic (tangential and normal centrifugal) and hydrodynamic (Archimedean screw and mobile phase drag) forces. By offering a set of quantitative or semi-quantitative theoretical frameworks, this work has resolved fundamental questions such as "in the absence of mobile phase flow, how is the distribution of the two phases in a CCC column determined?" and "for Type-J CCC, do the helical and the spiral columns lead to similar performance?"     

Role of counter-current chromatography in the modernisation of Chinese herbal medicines

This review focuses on the growing popularity of using counter-current chromatography (CCC), with its liquid stationary phase, as one of the prime methods for isolating compounds from Chinese herbal medicines (CHMs). 198 publications are reviewed covering 108 different plant species from 56 plant families. These describe the isolation of 354 different molecules across a wide range of polarities, chemical classes and molecular weights (in the range 100–1000 Da). The suitability of CCC for the separation of active compounds from CHM, the phase systems used, how CCC has developed in China, compounds isolated, CCC instrumentation, performance, operational issues and innovations, all supported by detailed cross-referencing, are described. It is concluded that CCC is making an increasingly important contribution to the modernisation of Chinese herbal medicines.     

New perspectives for microbial glycolipid fractionation and purification processes

Microbial glycolipids produced from renewable sources are of considerable interest in light of their promising biological activities and surfactant characteristics when compared to petroleum derived surfactants. Intense research efforts are currently being made to reduce their production costs and optimize recovery as selected mixtures through downstream processes. Due to the high complexity of natural glycolipid mixtures, efficient purification techniques are also required to examine the biological mechanisms of individual species towards human systems for their application in health-related areas. This review deals with recent advances in the development of glycolipid extraction, fractionation and purification methods, with a particular focus on solid support-free liquid-liquid separation techniques including centrifugal partition chromatography (CPC) and counter-current chromatography (CCC). These techniques offer promising perspectives for the preparative or large-scale separation of glycolipids from complex crude extracts, mainly because of their flexibility in solvent system selection and applicability to a diversity of structures of any polarity.     

Purification of high-throughput organic synthesis libraries by counter-current chromatography

Experiments were performed to evaluate whether counter-current chromatography (CCC) could function as an alternative purification method to reversed-phase high-performance liquid chromatography (RP-HPLC) and normal-phase supercritical fluid chromatography (SFC). RP-HPLC and SFC are the routine methods currently used in our high-throughput purification (HTP) facility for the purification of high-throughput organic synthesis (HTOS) libraries and medicinal chemistry reaction mixtures. Pre-equilibration of the solvent mixture layers was not mandatory for effective chromatography when hexanes–ethyl acetate–methanol–water (HEMW) solvent mixtures were used. Key to the use of CCC for high-throughput applications is the ability to effectively select a solvent system appropriate to each library member. Pilot-scale CCC elution time was used to estimate a starting solvent ratio and RP-HPLC retention time was then used to adjust solvent ratios within a particular library. It was also found that dimethyl sulfoxide (DMSO) and DMSO–methanol were suitable as sample injection solvents when using the HEMW solvent systems.     

Peak shape and dispersion behavior of solutes in counter-current chromatography with a single phase

The dispersion behavior of solutes was investigated in a rotating flowing coiled tube. Potassium iodide, tartrazine, ascorbic acid, lysozyme, bovine serum albumin (BSA), and silver nanoparticle (AgNPs) samples were eluted in a coiled tube of counter-current chromatography (CCC) apparatus with a single phase. Apparent convection peaks of low-diffusivity solutes appeared in the static CCC tube, while Gaussian-like peaks showed up for the high-diffusivity solutes. When the rotation speed of the CCC apparatus was elevated, all solute peak widths became smaller, and the convection peaks of AgNPs and BSA were minimized and formed Gaussian-like peaks. The axial dispersions of all solutes were reduced owing to the higher radial mass transfer in the rotating CCC column. The same reasoning could also be used to rationalize other special band shapes encountered in two-phase CCC separations.     

Enantioseparations in counter-current chromatography and centrifugal partition chromatography

Examples of chiral separations in counter-current chromatography (CCC) and centrifugal partition chromatography (CPC) are not numerous, due to the difficulty of finding chiral selectors highly selective in the liquid phase as well as a combination of solvents that does not destroy the selectivity and retains the capacity to elute chiral isomers of interest. New ideas and new chiral selectors generally come from other separation techniques, as will be highlighted in this review.     

Determination of the threshold odor concentration of main odorants in essential oils using gas chromatography-olfactometry incremental dilution technique

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.     

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.     

Separation and purification of alkaloids and phenolic acids from Phellodendron chinense by pH-zone refining and online-storage inner-recycling counter-current chromatography

In this work, eight compounds from Phellodendron chinense were separated and purified by pH-zone refining counter-current chromatography and traditional counter-current chromatography coupled with online-storage inner-recycling counter-current chromatography (IRCCC). The pH-zone-refining mode was adopted for separating 2.0 g of crude extract with the solvent system of chloroform–methanol–water (4:3:3, v/v), in which 10 mM hydrochloric acid and 10 mM triethylamine were added in the stationary and mobile phases, respectively. Meanwhile, traditional counter-current chromatography coupled with online-storage IRCCC separation was performed by the solvent system of n-hexane-ethyl acetate-methanol-water (5:5:2:8, v/v). Finally, eight compounds, including six alkaloids as 6-methylpiperidin-2-one( 1 ), isoplatydesmine( 4 ), berlambine( 5 ), epiberberine( 6 ), palmatine( 7 ), berberine( 8 ) and two phenolic acids as ferulic acid( 2 ), isoferulic acid( 3 ), were successfully obtained using these three different CCC modes with the purities over 95.0%.     

Application of preparative high-speed counter-current chromatography for separation of methyl gallate from Acer truncatum Bunge

Preparative separation of methyl gallate in leaves extract of Acer truncatum Bunge was conducted using high-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-ethanol-water at volume ratios of 5:1:5 (v/v/v). In a single operation, 57.5 mg of methyl gallate was obtained from 120 mg of the extract. HPLC analyses of the counter-current chromatography (CCC) fraction revealed that the methyl gallate was having over 97% purity. Its structure was identified by 1H NMR and 13C NMR.