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Copyright © 2021 The Author(s). Adenoviruses (AdVs) are used as gene therapy vectors to treat human diseases and as vaccines against COVID-19. AdVs are produced by transfecting human embryonic kidney 239 (HEK293) or PER.C6 virus producer cells with AdV plasmid vectors or infecting these cells withcell lysates containing replication-defective AdV. Cell lysates can be purified further by caesium chloride or chromatographic protocols to research virus seed stocks (RVSS) for characterisation to high quality master virus seed stocks (MVSS) and working virus seed stocks (WVSS) before downstream production of pure, high titre AdV. Lysates are poorly infectious, block filtration columns and have limited storage capability. Aqueous two-phase systems (ATPS) are an alternative method for AdV purification that rapidly generates cleaner RVSS for characterisation to MVSS. After testing multiple ATPS formulations, an aqueous mixture of 20 % PEG 600 and 20 % (NH4)2SO4 (w/w) was found most effective for AdV partitioning, producing up to 97+3% yield of high-titre virus that was devoid of aggregates both effective in vitro and in vivo with no observable cytotoxicity. Importantly, AdV preparations stored at −20 °C or 4 °C show negligible loss of titre and are suitable for downstream processing to clinical grade to support the need for AdV vaccines. Brunel University London Brief award.

Ampelozizyphus amazonicus Ducke (Rhamnaceae), a medicinal plant used to prevent malaria, is a climbing shrub, native to the Amazonian region, with jujubogenin glycoside saponins as main compounds. The crude extract of this plant is too complex for any kind of structural identification, and HPLC separation was not sufficient to resolve this issue. Therefore, the aim of this work was to obtain saponin enriched fractions from the bark ethanol extract by countercurrent chromatography (CCC) for further isolation and identification/characterisation of the major saponins by HPLC and MS. The butanol extract was fractionated by CCC with hexane - ethyl acetate - butanol - ethanol - water (1:6:1:1:6; v/v) solvent system yielding 4 group fractions. The collected fractions were analysed by UHPLC-HRMS (ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry) and MSn. Group 1 presented mainly oleane type saponins, and group 3 showed mainly jujubogenin glycosides, keto-dammarane type triterpene saponins and saponins with C31 skeleton. Thus, CCC separated saponins from the butanol-rich extract by skeleton type. A further purification of group 3 by CCC (ethyl acetate - ethanol - water (1:0.2:1; v/v)) and HPLC-RI was performed in order to obtain these unusual aglycones in pure form.

Phenylethanoid glycosides (PhGs) and iridoids are two vast groups of water soluble secondary metabolites widely distributed in plant kingdom and well known for their important biological activities. Their purification by conventional chromatography is time consuming, uses large amounts of organic solvents and requires repeated steps. Moreover, the sample recovery is low, because the hydroxyl groups of PhGs and iridoids make them strongly adsorbed onto the solid support (silica gel, Sephadex LH-20) during separation. Being a liquid–liquid based technology, countercurrent separation (CCS) comes as an alternative tool to overcome the issues associated with solid-phase adsorbents. It has been successfully used for the separation of many groups of specialized plant metabolites. This is the first extensive review describing the application of CCS for purification of iridoids and PhGs, based on the research papers from the last 20 years (1998–2018) that used countercurrent chromatography and centrifugal partition chromatography technologies. In total, 65 papers described the isolation of 84 different phytochemicals (28 PhGs and 56 iridoids) from 42 plant species belonging to 16 distinct families that were separated with 59 different biphasic solvent systems. Since PhGs and iridoids are highly polar molecules, more than half of the employed systems (56%) were composed of various ratios of ethyl acetate–alcohol–water, as well as binary systems, such as ethyl acetate–water and n-butanol– water. The current review may be used as starting point for CCS users on their sinuous road of isolating known or waiting to be discovered PhGs and iridoids.

The article reveals the influence of extracurricular projects in a foreign language on the students’ motivation in non-linguistic areas of university education for learning a foreign language. Extracurricular projects include Olympiads, conferences and various competitions for translation, essays, videos and similar types of extracurricular activities. The article reveals the illumination of the problem of motivation in the psychological and pedagogical community and gives various points of view on the factors that contribute to increasing motivation for learning a foreign language through extracurricular projects. A survey of students with non-linguistic specialization of the university revealed the key positive and negative factors that determine the attitude of students to such projects. The insignificant number of project participants is explained by the lack of awareness among students and fear of failure. The factors encouraging participation were the interest in the format of the event, the expected benefits for professional development, the support of the environment and a sense of belonging to the faculty. On the basis of the results obtained, recommendations are given to increase the motivation of students to learn a foreign language through participation in projects in foreign languages, and the need to integrate extracurricular academic activity into the educational program is substantiated.

Countercurrent chromatography (CCC) is being widely used across the world for purification of various materials, especially in natural product research. The predictability of CCC scale-up has been successfully demonstrated using specially designed instruments of the same manufacturer. The reality is that the most of CCC users do not have access to such instruments and do not have enough experience to transfer methods from one CCC column to another. This unique study of three international teams is based on innovative approach to simplify the scale-up between different CCC machines using fractionation of Schinus terebinthifolius berries dichloromethane extract as a case study. The optimized separation methodology, recently developed by the authors (Part I), was repeatedly performed on CCC columns of different design available at most research laboratories across the world. Hexane - ethyl acetate - methanol - water (6:1:6:1, v/v/v/v) was used as solvent system with masticadienonic and 3β-masticadienolic acids as target compounds to monitor stationary phase retention and calculate peak resolution. It has been demonstrated that volumetric, linear and length scale-up transfer factors based on column characteristics can be directly applied to different i.d., volume and length columns independently on instrument make in an intra-apparatus scale-up and inter-apparatus method transfer. F.N. Costa and S. Ignatova would like to thank Newton AdvancedFellowship project funded by the Royal Society of the United King-dom, which made this international work feasible. M.N. Vieirathanks the Studienstiftung desdeutschen Volkes (Germany) for thePh.D. scholarship. The authors are indebted to Plantextrakt GmbH& Co. (Germany) for the supply of S. terebinthifolius berry material.

This paper builds on the fact that high aspect ratio rectilinear tubing columns of the same length and outside dimensions can double column efficiency. It demonstrates that further improvements in efficiency can be made by using rectilinear tubing columns with half the wall thickness thus replacing heavy PTFE with light solvent systems and producing lighter higher capacity columns. Increases in sample loading/throughput of up to 55x are demonstrated by comparing the separation of Honokiol and Magnolol using a Hexane: Ethyl Acetate: Methanol: Water (5:2:5:2) phase system with the new thin wall rectilinear column (56 mL, 30 mL/min, 2.1 g/h in 6.5 min.) with the original optimization performed using a conventional DE-Mini column (18 mL, 0.8 mm bore circular PTFE tubing, 2.5 mL/min, 0.038 g/h in 45 min.). Honokiol is currently going through first phase clinical trials as an anti-lung cancer therapy where preparative countercurrent chromatography was used for its manufacture. To be competitive in the future it is important for the technology to become more efficient. This is the first big step in that direction.

Increasing column/tubing aspect ratio has been shown in a feasibility study to improve column efficiency when operating in reversed phase mode. This paper contains a thorough investigation on how increases in mobile phase flow and centrifugal force field affect stationary phase retention and column efficiency (as measured by the resolution between adjacent peaks) for columns wound with rectilinear tubing of different aspect ratio. The study uses a Mini CCC instrument operating from 1500 to 2100 rpm (126-246 g) to compare three columns with the same cross-sectional area but different aspect ratio - rectangular horizontal (force field perpendicular to the flat side - aspect ratio 3.125); square (aspect ratio 1.0) and rectangular vertical (flat side parallel with force field - aspect ratio 0.32). Columns are compared by measuring stationary phase retention, resolution and normalized resolution for 3 different mobile phase flow rates 2, 4 and 8 ml/min in both normal phase and reversed phase modes. The results with rectilinear tubing are compared to conventional circular tubing with the same cross-sectional area. The results show that resolution increases with aspect ratio and that at the highest aspect ratio the highest flow rate can maintain a high efficiency only if the highest g-field of 246 g is used. When comparing the rectangular horizontal tubing which gave the best results with conventional circular tubing with the same cross-sectional area a 45% improvement was found in reversed phase mode and a 51% improvement in normal phase mode over the conventional circular cross-section tubing. In other words, a rectangular horizontally wound bobbin with half the length of tubing can achieve the same result as a circular one. These are very significant results for halving separation times analytically or enabling designers to produce new instruments of the same capacity with a much-reduced size.

A robust strategy for the automated preparation of aqueous two-phase systems (ATPS) using a liquid handling sample processor was developed using gravimetric methods: to determine the accuracy of preparation. The major robotic control parameters requiring adjustment were; speed of aspiration and dispense; delay times following aspiration and dispense alongside measures to control cross-contamination during phase sampling. In general mixture compositions of both polymer/polymer and polymer/salt mixtures could be prepared with a target bias accuracy of less than 5%. However, we found that the bias accuracy with which systems of defined TLL and MR could be constructed was highly dependent on the tie line length of the ATPS and the geometrical form of the ATPS co-existence curve. For systems with a very low degree of curvature (PEG/salt systems here) increases in bias (accuracy) are appreciable at relatively long tie line lengths. Where the degree of curvature is more pronounced (PEG/dextran systems) closer approach to the critical point was possible without major effect on bias/accuracy. Application of the strategy to the measurement of the partitioning of phosphorylated and dephosphorylated forms of the model protein ovalbumin are reported. Differences in partition of phosphorylated (native) forms and dephosphorylated forms could be demonstrated. In a PEG/salt system this was manifest as a substantial decrease in solubility based on overall protein recovery derived from accurate knowledge of the system mass ratio. In a PEG/dextran system differences in partition coefficient could be demonstrated between phosphorylated and dephosphorylated forms.

The 8th International Conference on Counter-current Chromatography (CCC2014) was held at Brunel University London from July 23rd to 25th, 2014. It has been 14 years since Brunel hosted the first International Conference on CCC (CCC2000) at the beginning of the millennium and therefore, it was a good opportunity to review the progress of this emerging technology and particularly the impact it is having with industry today.

Countercurrent chromatography (CCC) is a form of liquid–liquid chromatography. It works by running one immiscible solvent (mobile phase) over another solvent (stationary phase) being held in a CCC column using centrifugal force. The concentration of compound in each phase is characterised by the partition coefficient ( K d ), which is the concentration in the stationary phase divided by the concentration in the mobile phase. When K d is between approximately 0.2 and 2, it is most likely that optimal separation will be achieved. Having the K d in this range allows the compound enough time in the column to be separated without resulting in a broad peak and long run time. In this paper we report the development of quantitative structure activity relationship (QSAR) models to predict log K d . The QSAR models use only the molecule's 2D structure to predict the molecular property log K d .

'Countercurrent chromatography' (CCC) is an ideal technique for the recovery, purification and isolation of bioactive natural products, due to the liquid nature of the stationary phase, process predictability and the possibility of scale-up from analytical to preparative scale. In this work, a method developed for the fractionation of Schinus terebinthifolius Raddi berries dichloromethane extract was thoroughly optimized to achieve maximal throughput with minimal solvent and time consumption per gram of processed crude extract, using analytical, semi-preparative and preparative 'high performance countercurrent chromatography' (HPCCC) instruments. The method using the biphasic solvent system composed of n-heptane-ethyl acetate-methanol-water (6:1:6:1, v/v/v/v) was volumetrically scaled up to increase sample throughput up to 120 times, while maintaining separation efficiency and time. As a fast and specific detection alternative, the fractions collected from the CCC-separations were injected to an 'atmospheric pressure chemical ionization mass-spectrometer' (APCI-MS/MS) and reconstituted molecular weight MS-chromatograms of the APCI-ionizable compounds from S. terebinthifolius were obtained. This procedure led to the direct isolation of tirucallane type triterpenes such as masticadienonic and 3β-masticadienolic acids. Also oleanonic and moronic acids have been identified for the first time in the species. In summary, this approach can be used for other CCC scale-up processes, enabling MS-target-guided isolation procedures.

For the provision of oleocanthal (OLC), a phenolic compound with very promising pharmacological properties, isolation from olive oil is a very important option. Due to the compound’s sensitivity to decomposition upon exposure to oxygen and light, a very gentle isolation method has been developed under use of high performance countercurrent chromatography (HPCCC). By partition of olive oil between hexane and methanol, an extract enriched in phenolics was prepared and subjected to a two-step HPCCC separation under use of heptane–EtOAc–MeOH–H2O mixtures in normal-phase and reverse phase mode, respectively. With this method, the isolation of tyrosol, hydroxytyrosol, and the mixture of (3S,4E)- and (3S,4Z)-OLC was achieved in approx. 70 min for each step. By one- and two-dimensional NMR-experiments and LC–MS, the equilibrium of (3S,4E)- and (3S,4Z)-OLC in such olive oil extracts has unambiguously been proven for the first time.

Betalains, natural plant pigments, are beneficial compounds due to their antioxidant and possible chemoprotective properties. A mixture of betalains: betanin/isobetanin, decarboxybetanins and neobetanin from processed red beet roots (Beta vulgaris L.) juice was separated in food-grade, gradient solvent systems using high-performance counter-current chromatography (HPCCC). The decarboxylated and dehydrogenated betanins were obtained by thermal degradation of betanin/isobetanin from processed B. vulgaris L. juice under mild conditions. Two solvent systems (differing in their composition by phosphoric acid and ethanol volume gradient) consisting of BuOH-EtOH-NaClsolution-H2O-H3PO4 (v/v/v/v/v, 1300:200-1000:1300:700:2.5-10) in the 'tail-to-head' mode were run. The flow rate of the mobile phase (organic phase) was 1.0 or 2.0 ml/min and the column rotation speed was 1,600 rpm (20°C). The retention of the solvent system stationary phase (aqueous phase) was ca. 80%. The system with the acid and ethanol volume gradient consisting of BuOH-EtOH-NaClsolution-H2O-H3PO4 (v/v/v/v/v, 1300:200-240:1300:700:2.5-4.5) pumped at 2.0 ml/min was the most effective for a separation of betanin/isobetanin, 17-decarboxy-betanin/-isobetanin, 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin pairs as well as neobetanin. The pigments were detected by LC-DAD and LC-MS. The results are crucial in the application of completely food-grade solvent systems in separation of food-grade compounds as well, and the systems can possibly be extended to other ionizable and polar compounds with potential health benefits. In particular, the method is applicable for the isolation and purification of betalains present in such rich sources as B. vulgaris L. roots as well as cacti fruits and Amaranthaceae flowering plants due to modification possibilities of the solvent systems polarity.

Over 8 million tonnes of sugar beet are grown annually in the UK. Sugar beet pulp (SBP) is the main by-product of sugar beet processing which is currently dried and sold as a low value animal feed. SBP is a rich source of carbohydrates, mainly in the form of cellulose and pectin, including d-glucose (Glu), l-arabinose (Ara) and d-galacturonic acid (GalAc). This work describes the technical feasibility of an integrated biorefinery concept for the fractionation of SBP and conversion of these monosaccharides into value-added products. SBP fractionation is initially carried out by steam explosion under mild conditions to yield soluble pectin and insoluble cellulose fractions. The cellulose is readily hydrolysed by cellulases to release Glu that can then be fermented by a commercial yeast strain to produce bioethanol at a high yield. The pectin fraction can be either fully hydrolysed, using physico-chemical methods, or selectively hydrolysed, using cloned arabinases and galacturonases, to yield Ara-rich and GalAc-rich streams. These monomers can be separated using either Centrifugal Partition Chromatography (CPC) or ultrafiltration into streams suitable for subsequent enzymatic upgrading. Building on our previous experience with transketolase (TK) and transaminase (TAm) enzymes, the conversion of Ara and GalAc into higher value products was explored. In particular the conversion of Ara into l-gluco-heptulose (GluHep), that has potential therapeutic applications in hypoglycaemia and cancer, using a mutant TK is described. Preliminary studies with TAm also suggest GluHep can be selectively aminated to the corresponding chiral aminopolyol. The current work is addressing the upgrading of the remaining SBP monomer, GalAc, and the modelling of the biorefinery concept to enable economic and Life Cycle Analysis (LCA).

International audience; Countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) are support free liquid-liquid chromatography techniques sharing the same basic principles and features. Method transfer has previously been demonstrated for both techniques but never from one to another. This study aimed to show such a feasibility using fractionation of Schinus terebinthifolius berries dichloromethane extract as a case study. Heptane - ethyl acetate - methanol -water (6:1:6:1, v/v/v/v) was used as solvent system with masticadienonic and 3β-masticadienolic acids as target compounds. The optimized separation methodology previously described in Part I and II, was scaled up from an analytical hydrodynamic CCC column (17.4mL) to preparative hydrostatic CPC instruments (250mL and 303mL) as a part of method development. Flow-rate and sample loading were further optimized on CPC. Mobile phase linear velocity is suggested as a transfer invariant parameter if the CPC column contains sufficient number of partition cells.

© 2017 American Chemical Society. A high-performance countercurrent chromatography (HPCCC) process was developed as a more efficient and sustainable alternative to reverse phase high-performance liquid chromatography (RP-HPLC) for the pilot-scale purification of the naturally occurring fermentation-derived insecticides, spinosyn A and spinosyn D, the major components of spinosad insecticide. While on pilot scale HPCCC and RP-HPLC both gave > 99% purities and comparable combined recoveries of 77% and 83%, respectively, HPCCC was much more efficient and sustainable by producing a 60% higher productivity, 11 times higher solute loading, 96% savings in stationary phase costs, and 42% reduction in solvent usage. The increase in productivity and reduction in solvent usage further reduced waste recycle and disposal costs, thus presenting significantly less environmental impact compared to RP-HPLC separations. The use of mixing on demand for a solvent system at the preparative scale allowed a complete automation with minimized solvent consumption.

Two mixtures of decarboxylated and dehydrogenated betacyanins from processed red beet roots (Beta vulgaris L.) juice were fractionated by high performance counter-current chromatography (HPCCC) producing a range of isolated components. Mixture 1 contained mainly betacyanins, 14,15-dehydro-betanin (neobetanin) and their decarboxylated derivatives while mixture 2 consisted of decarboxy- and dehydro-betacyanins. The products of mixture 1 arose during thermal degradation of betanin/isobetanin in mild conditions while the dehydro-betacyanins of mixture 2 appeared after longer heating of the juice from B. vulgaris L. Two solvent systems were found to be effective for the HPCCC. A highly polar, high salt concentration system of 1-PrOH-ACN-(NH4)2SO4 (satd. soln)-water (v/v/v/v, 1:0.5:1.2:1) (tail-to-head mode) enabled the purification of 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin and neobetanin (all from mixture 1) plus 17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2-decarboxy-neobetanin and 2,15,17-tridecarboxy-neobetanin (from mixture 2). The other solvent system included heptafluorobutyric acid (HFBA) as ion-pair reagent and consisted of tert-butyl methyl ether (TBME)-1-BuOH-ACN-water (acidified with 0.7% HFBA) (2:2:1:5, v/v/v/v) (head-to-tail mode). This system enabled the HPCCC purification of 2,17-bidecarboxy-betanin/-isobetanin and neobetanin (from mixture 1) plus 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2,17-bidecarboxy-2,3-dehydro-neobetanin and 2,15,17-tridecarboxy-neobetanin (mixture 2). The results of this research are crucial in finding effective isolation methods of betacyanins and their derivatives which are meaningful compounds due their colorant properties and potential health benefits regarding antioxidant and cancer prevention. The pigments were detected by LC-DAD and LC-MS/MS techniques.

This paper describes an equilibrium cell model for intermittent counter-current extraction that is analytically solved for the first time for continuous sample injection between a pair of columns. The model is compared with practice for injections of a model mixture of compounds on a standard high-performance counter-current chromatography instrument giving good agreement for compound elution order and the times to maximum concentration for the eluted components. An improved design of end fittings for the counter-current chromatography bobbins is described which permits on-column switching of the mobile and stationary phases. This on-column switching successfully eliminates the displaced stationary phase seen in fractions when operating ICcE with standard flying leads and gives a 6% reduction in the retention time of compounds and improved resolution due to the elimination of the time delay required to pump the previous mobile phase from standard flying leads.

The isolation of component sugars from biomass represents an important step in the bioprocessing of sustainable feedstocks such as sugar beet pulp. Centrifugal partition chromatography (CPC) is used here, as an alternative to multiple resin chromatography steps, to fractionate component monosaccharides from crude hydrolysed sugar beet pulp pectin. CPC separation of samples, prepared in the stationary phase, was carried out using an ethanol: ammonium sulphate (300gL

The need for new antimicrobial agents is greater than ever because of the emergence of multidrug resistance in common pathogens and incidence of new infections. Cyclopent-4-ene-1,3-diones (CPDs) have been reported as a new class of compounds with promising antimicrobial and antifungal properties. Herein we report the selective antibiotic properties of nostotrebin 6, a phenolic CPD produced biotechnologically by the culture of cyanobacterium Nostoc sp. str. Lukešová 27/97. High performance countercurrent chromatography (HPCCC) combined with gel permeation chromatography (GPC) was used for the isolation of nostotrebin 6 with a relatively high 0.53 ± 0.1% yield (calculated from dried biomass) and final purity higher than 96%. Nostotrebin 6 was tested for its antimicrobial and antifungal activities by using standard micro-dilution method, and the results were expressed as minimal inhibitory concentrations (MICs). Nostotrebin 6 unequivocally inhibited the growth of Gram-positive reference (Enterococcus faecalis CCM 4224, Staphylococcus aureus CCM 4223 and Staphylococcus aureus CCM 3953) and multidrug-resistant (Staphylococcus haemolyticus A/16568, Staphylococcus aureus MRSA 4591 and Enterococcus faecium VanA 419/ana) strains. Its strongest effect was exerted against the Gram-positive bacteria with MICs ranging between 6.25 and 15.6 μg/mL. There was no effect on Gram-negative strains tested and yeasts. Our results suggest that nostotrebin 6 could serve as basic nucleus for further design of novel antibiotic agents and demonstrate that the bio-production approach based on HPCCC/GPC isolation endpoint is an efficient methodology for obtaining nostotrebin 6 in multi-gram scale. Furthermore, the presented isolation method can be easily up-scaled to process kilograms of the cyanobacterial biomass. This work was supported by the Palacky University Internal Financial Support, project no. IGA_LF_2016_012 (J.V.) and IGA_LF_2016_022 (M.K.), by the project ALGAIN (CZ.1.07/2.3.00/30.0059, J.C.), by the Center for Algal Biotechnology-ALGATECH (CZ. 1.05/21.00/03.0110, J.C., J.K. and P.H.) and by the National Programme of Sustainability I, Ministry of Education Youth and Sports of the Czech Republic, ID: LO1416 (J.C., J.K. and P.H.).

Counter-current chromatography (CCC) has been widely used as a preparative separation method to purify natural products from plant extracts and fermentation broths. Traditionally, throughput optimization in CCC has focused on sample concentration and sample volume. In this paper sample injection was considered as consisting of three variables: injection flow rate, post-injection flow rate and sample solvent. The effects of these parameters were studied using a honokiol purification from a Magnolia officinalis bark extract as a case study aiming to achieve the highest throughput/yield ratio for greater than 99% purity of this potential anti- cancer drug obtained for submission to the Chinese FDA. An injection method was established that increased the throughput of honokiol by 46.5% (from 3.05 g/h to 4.47 g/h), and decreased the solvent consumption of mobile phase and stationary phase per gram of 35 honokiol by 40.0% (from 0.68 L/g to 0.41 L/g) and 48.4% (from 0.40 L/g to 0.21 L/g) 36 respectively. These results show the importance of understanding the whole injection process 37 when optimizing a given CCC separation. This research was supported by the National Natural Science Foundation of China (81302663), National High Technology Research and Development Program of China (863 Program) (2014AA020540) and 2011 Collaborative Innovation Program of China.

Countercurrent chromatography (CCC) is a developing chromatographic technique that achieves separation based on the distribution of the target in an immiscible biphasic or multiphasic solvent system. In the past decades, this technique has advanced greatly in application and theory. This overview is mainly focused on the development of elution modes, which have been easily achieved with the classical CCC apparatus in recent years. It includes gradient elution, dual-mode elution, multiple dual-mode elution, recycling elution, extrusion elution, cocurrent elution and pH zone refining. The basic principles of each elution mode are described in detail and then summarized. The comparison and the scope of application of these elution modes are also discussed. The financial support of the National Natural Science Foundation of China (NSFC No. 21175142 and No. 21505144) and Open Fund of Key Laboratory of Chemistry of Northwestern Plant Resources of the Chinese Academy of Sciences (No. CNPR-2011kfkt-02) is acknowledged. Dr X.-Y Huang is also grateful for the financial support from CAS to undertake overseas academic visits; he also acknowledges the Advanced Bioprocessing Centre at Brunel University, UK, for presenting the opportunity to study at Brunel

Gardenia jasminoides Ellis has been used for traditional Chinese medicine to treat all forms of febrile diseases. A cost-efficient and process-efficient separation of the bioactive molecule geniposide from G. jasminoides Ellis by high-performance counter-current chromatography (HPCCC) has been developed. Separation by preparative HPCCC was performed with a two-phase solvent system composed of ethyl acetate–butanol–water (0.4:1.6:2, v/v). The separation yielded 445 mg of geniposide with a purity of 95.2% from 4.0 g of crude extract. A factor of cost efficiency was developed and used for an economic evaluation of the separation process. The cost efficiency for geniposide was 0.070 g h −1 $ −1 . The separation process by HPCCC was demonstrated to be more economical and efficient than those methods by HSCCC and LC.

Ethnopharmacological relevance: Studies on traditional Chinese medicine (TCM), like those of other systems of traditional medicine (TM), are very variable in their quality, content and focus, resulting in issues around their acceptability to the global scientific community. In an attempt to address these issues, an European Union funded FP7 consortium, composed of both Chinese and European scientists and named "Good practice in traditional Chinese medicine" (GP-TCM), has devised a series of guidelines and technical notes to facilitate good practice in collecting, assessing and publishing TCM literature as well as highlighting the scope of information that should be in future publications on TMs. This paper summarises these guidelines, together with what has been learned through GP-TCM collaborations, focusing on some common problems and proposing solutions. The recommendations also provide a template for the evaluation of other types of traditional medicine such as Ayurveda, Kampo and Unani. Materials and methods: GP-TCM provided a means by which experts in different areas relating to TCM were able to collaborate in forming a literature review good practice panel which operated through e-mail exchanges, teleconferences and focused discussions at annual meetings. The panel involved coordinators and representatives of each GP-TCM work package (WP) with the latter managing the testing and refining of such guidelines within the context of their respective WPs and providing feedback. Results: A Good Practice Handbook for Scientific Publications on TCM was drafted during the three years of the consortium, showing the value of such networks. A "deliverable - central questions - labour division" model had been established to guide the literature evaluation studies of each WP. The model investigated various scoring systems and their ability to provide consistent and reliable semi-quantitative assessments of the literature, notably in respect of the botanical ingredients involved and the scientific quality of the work described. This resulted in the compilation of (i) a robust scoring system and (ii) a set of minimum standards for publishing in the herbal medicines field, based on an analysis of the main problems identified in published TCM literature. Conclusion: Good quality, peer-reviewed literature is crucial in maintaining the integrity and the reputation of the herbal scientific community and promoting good research in TCM. These guidelines provide a clear starting point for this important endeavour. They also provide a platform for adaptation, as appropriate, to other systems of traditional medicine. © 2012 Elsevier Ireland Ltd. All rights reserved., http://dx.doi.org/10.1016/j.jep.2012.01.038, SCOPUS: re.j, info:eu-repo/semantics/published

A vast majority Chinese herbal medicines (CHM) are traditionally administered as individually prepared water decoctions (tang) which are rather complicated in practice and their dry extracts show technological problems that hamper straight production of more convenient application forms. Modernised extraction procedures may overcome these difficulties but there is lack of clinical evidence supporting their therapeutic equivalence to traditional decoctions and their quality can often not solely be attributed to the single marker compounds that are usually used for chemical extract optimisation. As demonstrated by the example of the rather simple traditional TCM formula Danggui Buxue Tang, both the chemical composition and the biological activity of extracts resulting from traditional water decoction are influenced by details of the extraction procedure and especially involve pharmacokinetic synergism based on co-extraction. Hence, a more detailed knowledge about the traditional extracts’ chemical profiles and their impact on biological activity is desirable in order to allow the development of modernised extracts that factually contain the whole range of compounds relevant for the efficacy of the traditional application. We propose that these compounds can be identified by metabolomics based on comprehensive fingerprint analysis of different extracts with known biological activity. TCM offers a huge variety of traditional products of the same botanical origin but with distinct therapeutic properties, like differentially processed drugs and special daodi qualities. Through this variety, TCM gives an ideal field for the application of metabolomic techniques aiming at the identification of active constituents.

A strategy for the development of a green process using counter-current chromatography technology is presented in this paper. The strategy began with solvent system selection, followed by linear scale-up from an analytical to a preparative process with optimized operating parameters. A two-stage separation using a multi-injection method was performed with a solvent system of hexane–dichloromethane–methanol–water (4:0.75:4:1) for the 1st stage and a hexane–ethanol–water (4:2:2) for the 2nd stage. A 191.8 mg of tanshinone IIA was purified, with a 97% purity and 34.4% recovery and a 276.7 mg of cryptotanshinone was separated, with a 95% purity and 31.8% recovery from 2.1 g of crude extract. Process parameters (throughput, efficiency, environmental risk factor and general process evaluation) and mass factors (mass intensity, separation mass efficiency and greenness) of a target were developed for monitoring of the counter-current chromatography process.

Gradient elution in CCC is a powerful tool, which needs further systematic development to become robust and easy to use. The first attempt to build a correlation between gradient elution profile and distribution ratio (KD) values for model mixtures containing typical representatives of pharmaceutical compounds is presented in this paper. The three step estimation of the solvent system composition of a heptane-ethyl acetate-methanol-water (HEMWat) series is described. The estimation is based on simple measurements of initial and final stationary phase retention for gradient elution run, calculating gradient distribution ratio and correlating it with static KD against HEMWat number. © 2011 Elsevier B.V. All rights reserved.

The aim of this research is to compare two continuous extraction technologies, intermittent counter-current extraction (ICcE) and dual flow counter-current chromatography (DFCCC), in terms of loading and throughput using the GUESSmix, and show the advantages and disadvantages of the two methods. A model sample containing caffeine, vanillin, naringenin and carvone, with a total load of 11.2 g, was employed with a hexane–ethyl acetate–methanol–water (2:3:2:3) phase system to evaluate an ICcE method on a preparative (912 ml coil volume) DE-Midi instrument. While DFCCC was carried out on a specially designed preparative (561 ml coil volume) bobbin installed in a similar Midi instrument case. While similar throughputs of 7.8 g/h and 6.9 g/h were achieved for the ICcE and DFCCC methods respectively, ICcE was demonstrated to have a number of advantages over DFCCC.

Ginseng is a popular herb worldwide and has had varied uses in traditional Asian medicine for thousands of years. There are several different species of the herb, but all share the same constituents. Ginsenosides, the most extensively studied chemical components of ginseng, are generally considered to be one of the most important active ingredients of the plant. In this study, we have developed fast and efficient methodology for isolation of four known ginsenosides Rf, Rd, Re and Rb1 from Ginseng by high performance counter-current chromatography (HPCCC) coupled with evaporative light scattering detection (ELSD). The crude sample for HPCCC was purified firstly from a ginseng extraction using macroporous resin. The enriched saponin fraction (480 mg) was separated by using methylene chloride-methanol-5 mM aqueous ammonium acetate-isopropanol (6:2:4:3, v/v,) as the two-phase solvent system and yielded 10.7 mg of Rf, 11.0 mg of Rd, 13.4 mg of Re and 13.9 mg of Rb1. The purity of these ginsenosides was 99.2%, 88.3%, 93.7% and 91.8%, respectively assessed by HPLC-DAD-ELSD, and their structures were characterized by electrospray ionization mass spectrometry (ESI-MS) and compared with standards. Ammonium acetate was used to shorten the separation time and eliminate emulsification together with a flow step-gradient. The salt can be removed by re-dissolving the sample using acetone.

This paper describes how high-performance counter-current chromatography (HPCCC) was used strategically for the separation of Tripterygium wilfordii Hook. f. Due to the complexity of Chinese herbal medicines, the initial ethanol crude extract was fractionated into seven fractions using medium-pressure liquid chromatography (MPLC). One terpenoid (triptolide) and three alkaloids (peritassine A, wilforgine and wilforine) were further separated from one of the MPLC fractions. This fraction (1.25 g) yielded 8 mg of triptolide and 28 mg of peritassines A after one HPCCC column pass and 30 mg of wilforgine and 120 mg of wilforine after a second column pass with respective purities of 97%, 93.6%, 95.0% and 94.4%, which were determined by high-performance liquid chromatography (HPLC). This was a one-step HPCCC separation, using an n -hexane–ethyl acetate–methanol–water (4:5:4:5, v/v) solvent system, where increases in theoretical plates have been sacrificed in favour of increasing throughput. Structures were identified by electrospray ionization mass spectrometry (ESI-MS), 1 H nuclear magnetic resonance ( 1 H NMR) and 13 C nuclear magnetic resonance ( 13 C NMR). Comparison of three different modes of eluting compounds retained in the liquid stationary phase: elution extrusion; dual mode and simple pump-out showed that simply pumping out the column contents at high flow gave better resolution and was eight times faster than the other two well-utilised methods. Triptolide and peritassines A were isolated for the first time from Tripterygium wilfordii Hook. f.

A critical step in the bioprocessing of sustainable biomass feedstocks, such as sugar beet pulp (SBP), is the isolation of the component sugars from the hydrolysed polysaccharides. This facilitates their subsequent conversion into higher value chemicals and pharmaceutical intermediates. Separation methodologies such as centrifugal partition chromatography (CPC) offer an alternative to traditional resin-based chromatographic techniques for multicomponent sugar separations. Highly polar two-phase systems containing ethanol and aqueous ammonium sulphate are examined here for the separation of monosaccharides present in hydrolysed SBP pectin: l-rhamnose, l-arabinose, d-galactose and d-galacturonic acid. Dimethyl sulfoxide (DMSO) was selected as an effective phase system modifier improving monosaccharide separation. The best phase system identified was ethanol:DMSO:aqueous ammonium sulphate (300gL(-1)) (0.8:0.1:1.8, v:v:v) which enabled separation of the SBP monosaccharides by CPC (200mL column) in ascending mode (upper phase as mobile phase) with a mobile phase flow rate of 8mLmin(-1). A mixture containing all four monosaccharides (1.08g total sugars) in the proportions found in hydrolysed SBP was separated into three main fractions; a pure l-rhamnose fraction (90%), a mixed l-arabinose/d-galactose fraction and a pure d-galacturonic acid fraction (90%). The separation took less than 2h demonstrating that CPC is a promising technique for the separation of these sugars with potential for application within an integrated, whole crop biorefinery.

Considerable advances have been made in the last two years on the industrial scale‐up of countercurrent chromatography. This paper briefly reviews the scale‐up progress being made by three groups, two in France and one in the UK before giving details of advances being made at Brunel Institute for Bioengineering, Brunel University in the UK on the scale‐up of their J‐type centrifuges.

One of the major factors restricting the use of CCC as an analytical tool is the speed at which a separation may be conducted. This paper describes the phased development of a new low volume capacity Milli‐CCC device, which is as rapid as HPLC, achieving high resolutions in minutes as opposed to hours, with the capability of linking with a mass spectrometer (CCC/MS). The Milli‐CCC J‐type apparatus has gears enclosed in a lubricated case to minimize noise. Its volume with one coil mounted in a cantilever style is 4.6 mL with 2.5 m of 0.76 mm bore tubing. It can rotate at a maximum speed of 2100 rpm. Stationary phase retention factor higher than 60% could be obtained with 1500 rpm and 1 mL/min producing separation of compounds with K D distribution coefficient of 1 in less than 5 min. The connection to MS was straightforward.

This paper describes two simple tests that will characterize the hydrodynamics of a given phase system in a given CCC unit. Furthermore, it shows that, in practice, only one of these tests is necessary and the other can be predicted from the first. It goes on to show that retention information can be accurately obtained using a marker peak for the solvent front, so that both retention and resolution data can be collected in one simple test. While stationary phase retention (and, hence, peak elution) was found to behave quite predictably and could be modelled, resolution was not so predictable. Nevertheless, for analytical CCC, it was found that resolution of benzyl alcohol (BA) and p‐cresol (PC) only reduced from 2.3 to 1.4 as flow increased 7‐fold from 0.3 mL/min to 2 mL/min. Increasing the speed from 1000 to 2000 rpm (a 4× increase in “g” field) only increased the resolution a factor of 1.4×, whereas it was expected (from the number of mixing/settling cycles) to double. It is concluded that, fo...

This paper describes an investigation of organic phase retention and how it is affected by changing certain variables that are in the control of the user. A binary approach is taken in order to perform a quick evaluation of how key factors, like the addition of an extraction agent or a change of temperature, flow, tubing material, and bore affect stationary phase retention. Several different phase systems were studied in this way (decane–water, chloroform–water, methylisobutylketone–water, and carbon tetrachloride–water). The results show that retention is higher and more stable for larger bore tubing, and that at the analytical scale, there are threshold affects at low rotational speeds, which can create significant differences in retention behavior.

The effect of hydrodynamic parameters and the specific features of instrument design on the efficiency of substance separation in countercurrent liquid chromatography (CCC) was studied using a constant retention factor of the stationary phase in the column. The study was conducted with the separation of benzyl alcohol and p-cresol in a two-phase liquid system heptane–ethyl acetate–methanol–water (1.4 : 0.6 : 1 : 1) in as an example. It was shown that the peak resolution is improved with an increase in the rotational speed of the column and a decrease in the flow rate of the mobile phase. The best peak separation was attained using columns for which the ratio of the column rotation radius to the radius of column revolution was 0.615. It was shown that countercurrent chromatography allows the separation of substances with low partition constants (K < 1) in dilute solutions. The volume of the test sample may be up to 15% of the total volume of the chromatography column.

The effects of physicochemical properties of two-phase liquid systems (interfacial tension and differences in density and viscosity) on the retention of the stationary phase in the column were examined. These effects mainly determine the separation parameters of compounds. Extractant/decane–aqueous phase systems were used; their physicochemical properties changed both as a result of adding an extractant (di-(2-ethylhexyl) phosphoric acid, trioctylamine, or tributyl phosphate) to the organic solvent and because of a change in the composition of the aqueous phase. Aqueous ammonium sulfate of varying concentration was used as a mobile phase. It was shown that interfacial tension substantially affects the behavior of the systems under consideration. An increase in the ammonium sulfate concentration only slightly affects the retention factor of the stationary phase in the column. With a proper choice of the stationary phase, countercurrent chromatography can be used for the extraction of components from salt solutions of various concentrations.