Preparative enantioseparation of (±)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide by centrifugal partition chromatography

The racemic compound (±)-N-(3,4-cis-3-decyl-1,2,3,4-tetrahydrophenanthren-4-yl)-3,5-dinitrobenzamide ((±)-1), an analogue of increased lipophilicity of the chiral selector (CS) contained in the Whelk-O^® HPLC chiral stationary phase (CSP) has been resolved into its enantiomers by applying centrifugal partition chromatography (CPC). Considering the known enantioselectivity of the Whelk-O^® CS for naproxen, and the reciprocity concept in enantioseparation, (S)-naproxen related compounds were tested as CSs. In the search for an adequate solvent system, the partition behaviour of the two solutes, CS and racemate, has been studied using several biphasic solvent mixtures. The optimal CS concentration and sample loading capacity were determined in the chosen solvent system. The search for an appropriate CS and solvent system, the scale-up and optimization of the enantiomer separation by CPC, as well as the rationale for the unexpected elution order of enantiomers, are here described. The comparison of the preparative CPC separation achieved with that in HPLC, using a CSP containing an analogous CS, resulted favourable to the former in terms of loading capacity, solvent consumption and throughput.     

Preparative isolation and purification of sinomenine from Sinomenium acutum by centrifugal partition chromatography

Preparative centrifugal partition chromatography (CPC) was successfully carried out for the separation of sinomenine from the methanolic extract of Sinomenium acutum stems and rhizomes. The optimum two-phase solvent system of CPC was composed of n-hexane/ethyl acetate/methanol/water at a volume ratio of 1:6:2:8 (v/v/v/v) with 0.1% triethylamine (TEA). Preparative CPC yielded 44.3 mg of sinomenine from 400 mg of MeOH extract with a purity of 96.9%.     

Centrifugal partition chromatography – A review of recent applications and some classic references

Countercurrent chromatography, based on liquid–liquid partitioning, has many technological variants. One of them is centrifugal partition chromatography, introduced by Wataru Murayama and Kanichi Nunogaki in 1982. This technique, like other countercurrent chromatography techniques, is based on the phenomenon of liquid–liquid partitioning between two immiscible liquid phases that stay at equilibrium. But the significant difference between this technique and others is the retention mechanism of stationary phase. In the case of centrifugal partition chromatography, this mechanism is based on hydrostatic force, formed by the centrifugal field in the rotor in one‐axis centrifuge. Sometimes that allows more control of stationary phase, for example, when aqueous two‐phase and other difficult solvent systems are used. However, the efficiency of the separation in centrifugal partition chromatography is also affected by a variety of parameters dependent on the sample properties in the solvent system, physical properties of the solvent system, parameters of the instrument, and the method. This article includes also recent ideas for improvements to the technique and broadening its application (e.g., (multiple) dual‐mode or elution–extrusion procedure, pH‐zone‐refining centrifugal partition chromatography, ion‐exchange centrifugal partition chromatography, online and offline coupling of centrifugal partition chromatography).     

Effect of Coriolis force on counter-current chromatographic separation by centrifugal partition chromatography

The effect of Coriolis force on the counter-current chromatographic separation was studied using centrifugal partition chromatography (CPC) with four different two-phase solvent systems including n-hexane-acetonitrile (ACN); tert-butyl methyl ether (MtBE)-aqueous 0.1% trifluoroacetic acid (TFA) (1:1); MtBE-ACN-aqueous 0.1% TFA (2:2:3); and 12.5% (w/w) polyethylene glycol (PEG) 1000-12.5% (w/w) dibasic potassium phosphate. Each separation was performed by eluting either the upper phase in the ascending mode or the lower phase in the descending mode, each in clockwise (CW) and counterclockwise column rotation. Better partition efficiencies were attained by the CW rotation in both mobile phases in all the two-phase solvent systems examined. The mathematical analysis also revealed the Coriolis force works favorably under the CW column rotation for both mobile phases. The overall results demonstrated that the Coriolis force produces substantial effects on CPC separation in both organic-aqueous and aqueous-aqueous two-phase systems.     

高效逆流色谱法制备苏木中的氧化巴西木素

氧化巴西木素是苏木的主要化学成分之一,具有广泛的药理活性且常作为染色剂应用于各行各业。采用传统柱色谱法进行分离,不仅会造成色谱柱材料的污染,也会造成活性成分的损失。故采用高效逆流色谱(HPCCC)对苏木中的活性化合物氧化巴西木素进行分离纯化。苏木乙醇提取物经乙酸乙酯萃取后直接进行高效逆流色谱分离。首先利用基于薄层色谱的常用溶剂体系估算法和摇瓶法结合高效逆流色谱分析模式进行溶剂体系筛选。结果表明,最佳溶剂体系为氯仿-甲醇-水(4∶3∶2, v/v/v)。高效逆流色谱以反相模式为洗脱模式,主机转速为1600 r/min,流速为10 mL/min,分离温度为25 ℃,检测波长为285 nm,在氯仿-甲醇-水(4∶3∶2, v/v/v)溶剂体系下,从500 mg苏木乙酸乙酯萃取物中一次性分离制备得到15.2 mg纯度为95.6%的氧化巴西木素及一微量成分caesappanin C。采用高效逆流色谱分离制备氧化巴西木素,可避免苏木中的活性成分氧化巴西木素对色谱柱中的固体填充材料染色和难以洗脱等问题,并缩短分离纯化工作时间,提高工作效率。故可将高效逆流色谱应用到苏木中其他色素化合物或其他染料植物的分离制备工艺研究中。     

Intensified extraction of ionized natural products by ion pair centrifugal partition extraction

The potential of centrifugal partition extraction (CPE) combined with the ion-pair (IP) extraction mode to simultaneously extract and purify natural ionized saponins from licorice is presented in this work. The design of the instrument, a new laboratory-scale Fast Centrifugal Partition Extractor (FCPE300^®), has evolved from centrifugal partition chromatography (CPC) columns, but with less cells of larger volume. Some hydrodynamic characteristics of the FCPE300^® were highlighted by investigating the retention of the stationary phase under different flow rate conditions and for different biphasic solvent systems. A method based on the ion-pair extraction mode was developed to extract glycyrrhizin (GL), a biologically active ionic saponin naturally present in licorice (Glycyrrhiza glabra L., Fabaceae) roots. The extraction of GL was performed at a flow rate of 20 mL/min in the descending mode by using the biphasic solvent system ethyl acetate/n-butanol/water in the proportions 3/2/5 (v/v/v). Trioctylmethylammonium with chloride as a counter-ion (Al336^®) was used as the anion extractant in the organic stationary phase and iodide, with potassium as counter-ion, was used as the displacer in the aqueous mobile phase. From 20 g of a crude extract of licorice roots, 2.2 g of GL were recovered after 70 min, for a total process duration of 90 min. The combination of the centrifugal partition extractor with the ion-pair extraction mode (IP-CPE) offers promising perspectives for industrial applications in the field of natural product isolation or for the fractionation of natural complex mixtures.     

Purification of thonningianins A and B and four further derivatives from Thonningia sanguinea by one‐ and two‐dimensional centrifugal partition chromatography

Thonningia sanguinea is a parasitic herb widely used in traditional African medicine. Dihydrochalcone glucosides (unsubstituted, substituted with hexahydroxydiphenoyl or galloyl moieties) are the main constituents in the subaerial parts of this plant. In the present study, purification of the six major compounds from a methanol extract of the plant's subaerial parts was achieved by centrifugal partition chromatography. A first dimension centrifugal partition chromatography separation with the solvent system methyl tert‐butyl ether/1,2‐dimethoxyethane/water (1:2:1) in the ascending mode enabled the isolation of the two major bioactive compounds thonningianin A and B from 350 mg of methanol extract within only 16 min with respectable yields (25.7 and 21.1 mg), purities (87.1 and 85%), and recoveries (71.2 and 70.4%). Using a multiple heart‐cutting strategy, the remaining four major dihydrochalcone glucosides of the extract were further separated in a second dimension centrifugal partition chromatography with the solvent system ethyl acetate/1,2‐dimethoxyethane/water (2:1:1) in the descending mode with high purities (88.9–98.8%).     

Strategies of solvent system selection for the isolation of flavonoids by countercurrent chromatography

Flavonoids form a large class of important naturally occurring bioactive compounds. Their isolation and purification from natural sources can sometimes be very difficult and time‐consuming when traditional phytochemical techniques are used. Countercurrent chromatography (CCC), a support‐free liquid–liquid partition chromatography technique, is very useful for the isolation of polar compounds and its use is increasing in the natural products field. In this paper, we propose strategies of solvent system selection for the isolation of flavonoids by CCC, based on data from the literature, plus incorporation of own practical experiences. The selected references report the isolation of over 300 different flavonoid compounds from more than 100 plant species, using 40 different solvent systems, showing the versatility of this technique. The solvent system hexane‐ethylacetate‐methanol‐water is proposed as a starting point for the separation of samples containing free flavonoids, as it was cited in more than 60% of the papers. A “fine tuning” step is proposed at each level of this solvent family. Other modifications include exchanging the alcohol in the system as well as introducing a fifth solvent. The solvent system ethyl‐acetate‐butanol‐water is proposed as the starting point for glycosylated flavonoids. Other solvent systems are also discussed. The use of gradients is proposed for samples containing both free and glycosylated flavonoids, as the polarity window is larger in these cases. High‐speed countercurrent chromatography was used in 89% of the reviewed data.     

Some New Developments in Centrifugal Partiton Chromatography and Applications in the Separation of Natural Products

Abstract

A two-pump method for the control of stationary to mobile phase ratios in a cartridge centrifugal partition chromatography system is described. This cartridge CPC set-up and a rotating coil instrument are used in the separation of natural products from complex mixtures contained in crude plant extracts.     

Multiple dual-mode centrifugal partition chromatography, a semi-continuous development mode for routine laboratory-scale purifications

Nowadays, centrifugal partition chromatography (CPC) separations can be routinely achieved at the laboratory scale. The solvent system selection has been made easy, as generic sets of solvent systems are described in publications and books. This approach, however, generally reduces the scope of optimization strategies for two important parameters: selectivity and sample solubility. This can be very limiting for the preparative separation of structurally similar compounds. Multiple dual-mode (MDM) CPC has been developed to provide an easy-to-use alternative technique to circumvent this problem. A MDM separation consists of a succession of dual-mode runs (i.e. multiple inversion of stationary and mobile phase) that can only be achieved because both chromatographic phases are liquids. This original elution mode is thus a semi-continuous process with a classical sample injection and which only requires a single CPC column. Underlying mechanisms of MDM were studied using a model mixture of acenaphthylene and naphthalene. A mixture of two synthetic pairs of diastereomers was then successfully submitted to MDM CPC, in the framework of the synthesis of biologically active compounds.     

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).     

Extraction and separation of lactate dehydrogenase inhibitors from Poria cocos (Schw.) Wolf based on a hyphenated technique and in vitro methods

Stroke is one of the most common diseases worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke, with natural products considered a promising source of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Poria cocos . Five lactate dehydrogenase inhibitors were selected: dehydropachymic acid, pachymic acid, dehydrotrametenolic acid, trametenolic acid, and eburicoic acid. The inhibitors were extracted and isolated with purities of 96.75, 98.15, 97.25, 95.46, and 94.88%, respectively, by using a new “hyphenated” strategy of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography by a two‐phase solvent system of n‐hexane/ethyl acetate/ethanol/water at the volume ratio 0.965:1.000:0.936:0.826 v/v/v/v. The bioactivity of the isolated compounds was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay in PC12 cells. The results also showed that the hyphenated technique of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography was an efficient method for the continuous extraction and online isolation of chemical constituents from medicinal herbs. Furthermore, the research route based on the activity screening, extraction, separation, and activity verification of the compounds offered advantages of efficiency, orientation, and objectivity.     

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.     

Solanidine isolation from Solanum tuberosum by centrifugal partition chromatography

The aim of this investigation was the preparative isolation of solanidine (aglycone of the two main potato glycoalkaloids: α‐chaconine and α‐solanine) from fresh Solanum tuberosum (cv. Pompadour) material by implementing a new preparation scheme using centrifugal partition chromatography (CPC). A setup for obtaining solanidine by hydrolysis of the glycoalkaloids found in the skin and sprouts of S. tuberosum was first developed. Then its isolation was carried out by the development of CPC conditions: the solvent system used for separation was ethyl acetate/butanol/water in the ratio 42.5:7.5:50 v/v/v, 0.6 g of crude extract were separated with a 8 mL/min flow rate of mobile phase while rotating at 2500 rpm. A run yielded 98 mg of solanidine (86.7 % recovery from the crude extract) in a one‐step separation. The purity of the isolated solanidine was over 98%. Thus, CPC has proven to be the method of choice to get solanidine of very high purity from S. tuberosum biomass in large quantities.     

New advances in countercurrent chromatography and centrifugal partition chromatography: focus on coupling strategy

Countercurrent chromatography (CCC) is an attractive separation method because the analytes are partitioned between two immiscible liquid phases avoiding problems related to solid stationary phase. In recent years, this technique has made great progress in separation power and detection potential. This review describes coupling strategies involving high speed CCC (HSCCC) or centrifugal partition chromatography (CPC). It includes on-line extraction–isolation, hyphenation with mass spectrometry (MS) and nuclear magnetic resonance (NMR) detectors, multidimensional CCC (MDCCC), two-dimensional CCC (2D-CCC), on-line coupling with liquid chromatography (LC), and biological tests, and innovative off-line developments. The basic principles of each method are presented and applications are summarized.     

Bioassay-Guided Isolation of Two Eudesmane Sesquiterpenes from Lindera strychnifolia Using Centrifugal Partition Chromatography

In this study, a centrifugal partition chromatography (CPC) separation was applied to identify antioxidant-responsive element (ARE) induction molecules from the crude extract of Lindera strychnifolia roots. CPC was operated with a two-phase solvent system composed of n-hexane-methanol-water (10:8.5:1.5, v/v/v) in dual mode (descending to ascending), which provided a high recovery rate (>95.5%) with high resolution. Then, ARE induction activity of obtained CPC fractions was examined in ARE-transfected HepG2 cells according to the weight ratios of the obtained fractions. The fraction exhibiting ARE-inducing activity was further purified by preparative HPLC that led to isolation of two eudesmane type sesquiterpenes as active compounds. The chemical structures were elucidated as linderolide U (1) and a new sesquiterpene named as linderolide V (2) by spectroscopic data. Further bioactivity test demonstrated that compounds 1 and 2 enhanced ARE activity by 22.4-fold and 7.6-fold, respectively, at 100 μM concentration while 5 μM of sulforaphane induced ARE activity 24.8-fold compared to the control.     

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.     

Separation and isolation of major polyphenols from maritime pine (Pinus pinaster) knots by two‐step centrifugal partition chromatography monitored by LC‐MS and NMR spectroscopy

Pine knots are a rich source of lignans, flavonoids, and stilbenes. These bioactive compounds are widely known for their roles to combat human disorders but also to protect plants against pathogens. In order to gain knowledge inside their potential activities, a suitable isolation and purification of these high‐added value compounds is required. To this end, centrifugal partition chromatography, as a rapid and useful methodology of separation, was employed and developed. The coefficient partition values (K_D) of six major compounds in nine biphasic solvent systems were determined to evaluate the most appropriate system. Two‐step centrifugal partition chromatography was required to separate lignans using ARIZONA system K (n‐heptane/ethyl acetate/methanol/water 1:2:1:2, v:v) and to isolate stilbenes and flavonoids using ARIZONA system P (n‐heptane/ethyl acetate/methanol/water 6:5:6:5, v:v). Eight one‐compound enriched‐fractions were obtained as follows: nortrachelogenin (70.1%), secoisolariciresinol (53.7%), isolariciresinol (61.1%), taxifolin (48.4%), pinocembrin (91.3%), pinobanksin (91.1%), pinosylvin (91.4%), and pinosylvin monomethyl ether (91.1%). Additionally, the centrifugal partition chromatography allowed to unravel the composition of pine knot owing to the several fractions generated. Twenty‐two compounds were characterized by liquid chromatography‐mass spectrometry and NMR spectroscopy, some of which are described for the first time in literature.     

Mutual Separation of Lanthanoid Elements by Centrifugal Partition Chromatography

Abstract

Multistage separation based on liquid-liquid extraction has been investigated by means of centrifugal partition chromatography (CPC). A kerosene solution of 2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester (EHPA) was employed as a stationary phase without any solid support. Metal ions eluted by the aqueous mobile phase were detected by the post-column reaction with Arsenazo III. The retention volumes are approximately linear with the distribution ratios of metals. The mutual separation of adjacent lanthanoids was accomplished by CPC.     

Scale-up of protein purifications using aqueous two-phase systems: comparing multilayer toroidal coil chromatography with centrifugal partition chromatography

Two different laboratory scale liquid-liquid extraction processes using aqueous two-phase systems (ATPS) are compared: centrifugal partition chromatography (CPC) and multilayer toroidal coil chromatography (MTCC). Both use the same phase system, 12.5% (w/w) PEG-1000:12.5% (w/w) K(2)HPO(4), the same flow rate of 10 mL/min and a similar mean acceleration field of between 220 × g and 240 × g. The main performance difference between the two processes is that there is a continuous loss of stationary phase with CPC, while for MTCC there is not - even when sample loading is increased. Comparable separation efficiency is demonstrated using a mixture of lysozyme and myoglobin. A throughput of 0.14 g/h is possible with CPC despite having to refill the system with stationary phase before each injection. A higher throughput of 0.67 g/h is demonstrated with MTCC mainly due to its ability to tolerate serial sample injections which significantly reduces its cycle time. While CPC has already demonstrated that it can be scaled to pilot scale, MTCC has still to achieve this goal.