new advances in countercurrent chromatography and centrifugal partition chromatography ... ›...

REVIEW

New advances in countercurrent chromatography and centrifugalpartition chromatography: focus on coupling strategy

Thomas Michel & Emilie Destandau & Claire Elfakir

Received: 25 March 2013 /Accepted: 23 April 2013 /Published online: 8 June 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Countercurrent chromatography (CCC) is an at-tractive separation method because the analytes arepartitioned between two immiscible liquid phases avoidingproblems related to solid stationary phase. In recent years,this technique has made great progress in separation powerand detection potential. This review describes couplingstrategies involving high speed CCC (HSCCC) or centrifu-gal partition chromatography (CPC). It includes on-lineextraction–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 develop-ments. The basic principles of each method are presentedand applications are summarized.

Keywords HSCCC . CPC .MS . On-line and off-linecoupling . Extraction . Purification . Natural products

Introduction

Countercurrent chromatography (CCC) is a generic termcovering all forms of liquid chromatography (LC) that usetwo immiscible liquid phases without any solid support [1].The liquid stationary phase is held in place by centrifugalforce while the mobile phase is pumped through it. Since itsinvention by Yoichiro Ito many developments have beenproposed. Modern CCC apparatus can be divided in twomain categories, the hydrodynamic and hydrostatic

equilibrium systems. The hydrodynamic system has tworotational axes, a main axis and planetary motion whichgenerates a variable centrifugal force field applied to con-tinuous open tubing coiled on bobbins. Today this techniqueis known as high-speed CCC (HSCCC) [2]. Use of thehydrostatic system is specific to centrifugal partition chro-matography (CPC) in which the column is characterized bysmall elution chambers interconnected by capillary tubing,with rotation around a single axis only [3]. Because of theuse of a liquid stationary phase, CCC benefits from manyadvantages over traditional preparative LC for example:

1. absence of irreversible adsorption of molecules;2. high loading capacity;3. total recovery of injected sample;4. low risk of sample denaturation;5. minimized tailing; and6. low solvent consumption [4, 5].

In addition, CCC is a versatile chromatographic tech-nique because of its ability to reverse the flow directionand interchange the mobile and stationary phases during agiven chromatographic process. Consequently the method isideal for preparative separation of natural products fromcrude extracts. In recent decades, many papers and reviewarticles have been published on separation of natural prod-ucts by CCC [4, 6–9]. It is obvious from the extensive recentliterature on CCC developments and applications that CCCis a promising preparative technique. However, it worthmentioning that CCC also has disadvantages, for examplelow efficiency (small number of theoretical plates), neces-sary optimisation of the separation conditions for each newsample, and requirement of a good sealing system to preventsolvent and sample leaks when the column is under rotation.

In recent years, multidimensional chromatography andon-line coupling strategies have emerged to enhance sepa-ration efficiency and peak capacity. On the analytical scale,hyphenation of high-performance liquid chromatography(HPLC) with different kind of detector, for example the

Published in the special issue Analytical Science in France with guesteditors Christian Rolando and Philippe Garrigues.

T. Michel : E. Destandau (*) : C. ElfakirUniversité d’Orléans‐CNRS, Institut de Chimie Organiqueet Analytique UMR 7311, BP 6759 rue de Chartres,45067 Orléans cedex 2, Francee-mail: [email protected]

Anal Bioanal Chem (2014) 406:957–969DOI 10.1007/s00216-013-7017-8

diode array detector (DAD), the evaporative light scatteringdetector (ELSD), mass spectrometry (MS), and nuclearmagnetic resonance (NMR) provides powerful techniquesfor compound identification [10–13]. Moreover a variety ofon-line chromatographic coupling techniques have beenimplemented, for example two-dimensional LC using twoorthogonal chromatographic separations to improve separa-tion of compounds reducing their coelution.

The main purpose of this review is to describe the state-of-art of on-line coupling strategy involving semi-preparative-scale countercurrent separation. Recent trendsinclude:

1. coupling of specific detectors to extend detection andcharacterization possibilities;

2. development of innovative hyphenation methods to im-prove efficiency and peak capacity of CCC; and

3. minimization of sample preparation and post-separationsteps to save time and operation cost in the wholepurification process.

For this purpose, different approaches involving hyphen-ation with MS and NMR detectors, multidimensional CCC(MDCCC), two-dimensional CCC (2D CCC), on-line cou-pling with HPLC or with biological evaluation, on-lineextraction–isolation, and multichannel separation will bedescribed.

Overview on CCC hyphenation with specific detectors

According to the literature, CCC has been coupled to dif-ferent detectors for on-line monitoring of the separationprocess. The most widespread detector is the non-destructive UV–visible detector which enables continuousmonitoring of fractionation steps and affords total recoveryof molecules. However, the systematic use of UV–visible isproblematic for isolation of compounds lacking a UV–vis-ible chromophore. The evaporative light scattering detector(ELSD) enables detection of such compounds and has,consequently, been widely used for detection of a varietyof analytes in CCC [14–17]. It is, furthermore, very useful inCCC because it evaporates the mobile phase, thus eliminat-ing baseline disturbance caused by stationary phase bleed-ing generally observed with an UV detector.

On the other hand, in combination with liquid chroma-tography or gas chromatography, MS has become a high-throughput technique in the fields of natural products andanalytical chemistry because of its sensitivity and its abilityto give immediate structural information without isolationprocesses. Overall, coupling of CCC with MS detectionenables rapid, simple, effective, and sensitive monitoringof purification. Indeed, the coupling of HSCCC with massspectrometry avoids the solvent problems (e.g. bleeding of

the stationary phase) associated with conventional UV de-tectors, and mass spectrometry enables on-line universaldetection with high sensitivity. Nevertheless, implementa-tion of this on-line procedure requires use of a volatilesolvent system for MS detection and a splitter system en-abling division and transfer of the fast flow stream from theCCC outlet into the mass spectrometer. Table 1 and the nextsection give an overview of 20 years of original researchwhich has made possible direct coupling of CCC or CPCwith MS.

CCC–MS on-line coupling

In the first development of CCC–MS instrumentation,reported in 1988 and 1990 by Lee et al. [18, 19], CCCwas directly interfaced with thermospray MS for iden-tification of alkaloids and lignans from plant extracts.Other MS ionisation techniques, including continuous-f low fast -atom bombardment (FAB), chemicalionisation (CI), electron ionisation (EI) [20], and FABvia a moving belt interface [21], have been coupledwith HSCCC via a T-split. Although effective, theseapproaches all suffer from a high back pressure gener-ated by the direct interface between chromatograph andmass spectrometer, which can damage the CCC columnand connection tubing and disturb the MS source. Toovercome this problem, HSCCC has been combinedwith reduced-pressure ionisation techniques, for exam-ple electrospray ionisation (ESI) and atmospheric-pressure chemical ionisation (APCI), which enabledapplication of HSCCC–MS to a variety of samples.

The effluent from CCC was divided, by use of a T-split,and transferred to a fraction collector and the MS instru-ment. Indeed, the use of a suitable flow-splitter is essential,because it enables reduction of flow streams and sampleamount directed to the mass spectrometer source. A typicalset-up of HSCCC–MS is illustrated in Fig. 1. HSCCC flowwas well divided because of the flow splitter; the main partis used for fraction collection whereas the smaller part isdirected to the mass spectrometer source for effluent mon-itoring and compound identification.

This approach has been widely used for separation andidentification of agrochemical compounds [22, 23], thera-peutic molecules [24–28], and natural products [29–34]. Forinstance, Inoue et al. applied HSCCC–ESI–MS to on-linepurification of avermectins, gentamicin, and polypeptideantibiotics [24–26]. Besides the T-split, their instrumentalset-up also used a micro splitter valve before the MSdetector to obtain a stable flow rate at 200 μL min−1

for ESI. The authors were able to detect and purifytarget compounds by use of the extracted ion modeeven if the two-phase solvent system comprised an

958 T. Michel et al.

apolar solvent, for example n-hexane, and ethyl acetate.Figure 2 demonstrates well that HSCCC–ESI–MS can be usedfor efficient on-line monitoring of such compounds as genta-micin, which do not have a chromophore.

Winterhalter’s group has developed preparative HSCCC–ESI–MSn well for target-guided fractionation and isolationof natural products. MS–MS data available throughout theentire chromatographic separation provide important infor-mation for elucidation of the structures of phytochemicals.They used this approach for fast screening and fractionation

of polyphenols from Hippophaë rhamnoides juice [30]. It isworth noting that they evaluated with success different splitconfigurations—a T-split and a Y-split which enabledivision of the HSCCC effluent stream by a ratio1:20 and 1:125, respectively. More recently, they usedpreparative ion-pair HSCCC–ESI–MSn for the target-guided separation of betacyanins from the bracts ofBougainvillea glabra [34] and HSCCC–MSn equippedwith an ESI and APCI sources for fractionation andisolation of acylated flavonoid glycosides and acardic

Table 1 Applications of on-line CCC–MS approach

Couplingtechnique

Ionisationsource

Molecule Sample Solvent system (v/v) Ref.

CCC–MS Thermospray Alkaloids Vinca minor – [18]

CCC–MS Thermospray Lignans Schisandra rubiflora n-Hexane–ethanol–water (6:6:5) [19]

Milli-CCC–MS

– Uracil, benzylalcohol,paracresol

– Heptane–ethyl acetate– methanol–water(1.4:0.6:1.0:1.0)

[38]

Dual CCC–MS–MS

ESI+ Carbamatepesticides

Mandarin orange andspinach samples

n-Hexane–acetonitrile–0.1 % formic acid (45:45:10) [22]

HSCCC–MS Moving-beltinterface FAB−

[21]

HSCCC–MS APCI− Barbiturates Chemical supplier Butyronitrile–acetonitrile–water (1:1:1) [27]

HSCCC–MS ESI− Pesticides Chemical supplier n-Hexane–ethyl acetate–methanol–water (1.4:1:1:1) [23]

HSCCC–MS EI, CI, and FAB Indole auxins Pharmaceuticalmaterial

n-Hexane–ethyl acetate–methanol–water (1:1:1:1) [20]Mycinamicins n-Hexane–ethyl acetate–methanol–8 % aqueous

ammonia (1:1:1:1)Colistin complex

n-butanol–0.04 mol L−1 aqueous TFA solution (1:1)containing 1 % glycerol

HSCCC–MSn

ESI Coumarins Peucedanumpraeruptorum

Light petroleum–ethyl acetate–methanol–water(5:5:6:4)

[31]

HSCCC–MS ESI+ Erythromycins Chemical supplier n-Hexane–ethyl acetate–methanol–water (4:7:4:3) [28]Didemnins Trididemnum soliduni n-Hexane–ethyl acetate–methanol–water (1:4:1:4)

HSCCC–MS ESI and APCI− Flavonoids Oroxylum indicum n-Hexane–ethyl acetate–methanol–0.2 % formic acid(1:1.2:1:1)

[29]

HSCCC–MS ESI+ Polypeptideantibiotics

Pharmaceuticalmaterial

n-Butanol–n-hexane–0.05 % aqueous trifluoroaceticacid solution (43:7:50)

[24]

HSCCC–MS ESI+ Gentamicincomponents

Gentamicin n-Butanol–10 % aqueous ammonia solution (1:1) [26]

HSCCC–MS ESI+ Avermectinisomers

Pharmaceuticalmaterial

n-Hexane–ethyl acetate–methanol–0.5 % formic acidin water (7:3:5:5)

[25]

HSCCC–MSn

ESI− Polyphenols Hippophaërhamnoides

n-Hexane–butanol–water (1:1:2) [30]

HSCCC–MSn

ESI Flavonoids Hippophaërhamnoides

n-Hexane–butanol–water (1:1:2) [33]

prepHSCCC–MSn

ESI and APCI Phenolic lipids Anacardiumoccidentale

n-Hexane–acetonitrile (1:1) [32]

IP–HSCCC–MSn

ESI+ Betacyanins Bougainvillea glabra tert-Butyl methyl ether–n-butanol–acetonitrile–water(2:2:1:5) containing trifluoroacetic acid

[34]

CPC–MS ESI− Flavonols Malus sp Ethyl acetate–ethanol–water (4.5:1:4.5) [36]

CPC–MS ESI+ and − Xanthones Garcinia mangostana Heptane–ethyl acetate–methanol–water (2:1:2:1) [35]

CPC–MS ESI Stilbenoids Vitis riparia × Vitisberlandieri

Heptane–ethyl acetate–methanol–water (1:2:1:2) [37]

HSCCC, high-speed countercurrent chromatography; CPC, centrifugal partition chromatography; MS, mass spectrometry; ESI, electrospray ionisation;APCI, atmospheric pressure chemical ionisation; EI, electron ionisation; CI, chemical ionisation; FAB, fast-atom bombardment; IP, ion-pair

New advances in CCC and CPC coupling 959

acid from Hippophaë rhamnoides and cashew nuts,respectively [32, 33].

CPC-MS on-line coupling

In contrast with HSCCC–MS, the on-line coupling of CPCwith MS is an emerging technique with the first reportspublished by our group in 2009. Destandau et al. [35]investigated the benefit of an active flow splitter to over-come problems linked with the connection of CPC and MS.In their study, the authors compared passive T-split andactive splitter (variable flow splitter VFS) for rapid screen-ing and fractionation of xanthones from Garciniamangostana. As depicted in Fig. 3, a variable flow-splitter(VFS) was used to enable discontinuous transfer of smallaliquots from the CPC effluent (100 nL) at 1.667 Hz throughan independent secondary auxiliary stream directed to themass spectrometer. The auxiliary flow, composed of1 mol L−1 ammonium acetate–ethanol (5:95, v/v), was de-livered by a secondary pump. Passive T-split limits separa-tion development because compounds must be eluted only

Fig. 1 Schematic diagramof the HSCCC–ESI–MS set-upof Inoue et al. Reprintedfrom Ref. [24], withpermission

Fig. 2 Total ion chromatogram (TIC) and extract ion chromatogramsof gentamicin compounds obtained by use of HSCCC–ESI–MS inpositive mode. The two-phase solvent system was n-butanol–10 %aqueous ammonia solution (1:1, v/v). Reprinted, with permission, fromRef. [26]


with aqueous mobile phase to be ionised in the MS, where-as, active split enables ionisation of compounds in bothaqueous and organic phase, enabling the development dif-ferent modes of separation (e.g. dual mode, elution–extru-sion) which reduce analysis time, as illustrated inFig. 4. However, it results in lower sensitivity and aless stable signal because of its discontinuous CPC flowstream transfer.

The same research team has further optimised CPC–ESI–MS for fractionation and isolation of glycoside flavonolsfrom an apple extract [36]. CPC–ESI–MS was configuredby coupling both instruments via the VFS and enabled directisolation, monitoring, and identification of flavonols.Compound structures were confirmed on-line by MS–MS in multiple reaction monitoring (MRM) mode. Fur-thermore, repeatable and reproducible VFS performanceenabled evaluation of the quantification potential of theCPC–ESI–MS approach.

Another French group recently interfaced semi-preparative CPC to an ESI ion-trap spectrometer by use ofan active splitter [37]. They also developed an innovative“back-step” elution mode to optimize difficult separationoccurring with isocratic CPC separation. The “back-step”elution mode consists in a step gradient mode from higher tolower eluent strength enabling better separation of targetcompounds from impurities. By use of this procedure, threehydroxystilbenes were purified from an extract of the root ofVitis riparia × Vitis berlandieri grapevine.

CPC–NMR coupling

Development of CPC–NMR can be an interesting tool forphytochemists. It combines the separation efficiency of CPCon the preparative scale with the structure-elucidation powerof NMR. The NMR was used to monitor CPC separation ofa test mixture of three N-2,4-dinitrophenyl amino acidsperformed in pH-zone-refining elution mode [39]. In thisstudy, experiments were realised in stop-flow mode whichenabled droplets from stationary phase to decant partiallyaway from the NMR observation zone. Spectra were ac-quired every 3 min between each CPC stop and with auto-matic solvent suppression. Here, it is worth mentioning thatalthough CPC–NMR was forgotten for a long time, thereseems to be renewed interest since Bisson et al. presentedtheir results in 2012 during International Congress on Nat-ural Products Research [40].

CPC–gustatometry and Fourier Transform MS

Recently an innovative off-line CPC–gustatometry hyphen-ation technique was implemented by Marchal et al. [41] forisolation of sapid compounds from an oak extract. CPCenables fractionation of the crude extract and then locationof the targeted compounds; all the fractions were tasted andtheir sweetness intensity was evaluated. Compounds werefurther identified by a combination of FTMS and 2D NMR.

CPC

Fraction collection

Mass spectrometer

Injection loop

ANALYSIS STREAM

SEPARATION STREAM

Auxillary pump

Primary pump

Active flow splitter

VFS

Fig. 3 Schematic diagram ofthe CPC–ESI–MS approach.The variable flow splitter (VFS)transfers a small volume of theCPC effluent to the massspectrometer at a frequency of1.667 Hz

50.00 100.00 150.00Time-1

100

%

2

3

5

4

x

67

100.00 200.00 300.00Time-1

100

%

2

3

5

4x

67

Dual mode

a bFig. 4 CPC–ESI–MSchromatograms of xanthonesfrom G. mangostana obtainedin negative ion mode with apassive splitter (a) and an activesplitter (b). Single-ionmonitoring (SIM) of m/z 379,385, 407, 409, 423 and 427.Reprinted from Ref. [35], withpermission


Trends in multidimensional techniques connectedto CCC

Multidimensional chromatography is based on the combi-nation of different techniques involving a variety of separa-tion mechanisms. In a typical multidimensional separation, asample is first subjected to separation by one method, thenthe separated components are further separated by at leastone additional independent method [42]. Hence, two-dimensional (2D) or multidimensional chromatography isgenerally characterized by enhanced resolution and peakcapacity over one-dimensional chromatography.

Multidimensional CCC (MDCCC)

In the last decade, multidimensional CCC (MDCCC) hasemerged as a powerful tool for separation of complex matri-ces, for example plant extracts. Overall, MDCCC usesswitching valves to interface the first column with the second,which results in more complex instrumentation. Establish-ment of a multidimensional separation also requires the useof multiple LC pumps, automated switching valves, detectors,and fraction collectors. The first to report development of anMDCCC system was Ito’s group in 1998 with the associationof two identical HSCCC instruments each equipped with a110 m×1.6 mm i.d. polytetrafluoroethylene (PTFE) multilay-er coil [43]. As depicted in Fig. 5, the heart of the systemcomprised two switching valves—one used for sample injec-tion and the other to transfer the sample into the secondcolumn. A typical separation procedure implies first solventfilling and equilibrium of the two columns. Sample is theninjected via the first six-port valve into column 1 while pump2 is stopped (configuration A, Fig. 5). When a target peakappears, the effluent from HSCCC 1 is introduced to HSCCC2 by use of the second valve (configuration B, Fig. 5). Thevalve is then returned to its first position while pump 2 isfinally restarted to separate the compound of interest withHSCCC 2. The MDCCC approach increases resolution andpeak capacity which enables separation of overlapped

compounds in a reduced separation time and with higherpurity. Consequently this methodology has great potentialfor isolation and purification of molecules with similar struc-tures from complex matrices, for example plant extracts.

MDCCC has been successfully used for separation ofdifferent natural products from Chinese medicinal herbs,as illustrated in Fig. 6. The method was first applied to thepreparative separation of flavonoids from Ginkgo bilobaand Hippophaë rhamnoides with a two-phase solvent sys-tem composed of chloroform–methanol–water (4:3:2, v/v)[43]. More recently, furanocoumarins from Angelicadahurica have been purified by use of a pair of two-phasesolvent systems composed of n-hexane–ethyl acetate–meth-anol–water at volume ratios of 1:1:1:1 (v/v) and 5:5:4.5:5.5(v/v) [44]. Tripdiolides from Tripterygium wilfordii weresimilarly isolated by use of n-hexane–dichloromethane–methanol–water (3:22:17:8, v/v) and chloroform–metha-nol–water (4:3:2, v/v) as the pair of two-phase solventsystems [45]. MDCCC was also used for isolation andpurification of tanshinones from the roots of Salviamiltiorrhiza by use of a combination of light petroleum–ethyl acetate–methanol–water in different proportions [46].

On the basis of this work, Pan and co-workers elaborateda more complex type of MDCCC they called two-dimensional CCC (2D-CCC) [47]. In this approach, theyused HSCCC columns of different volumes in the first andsecond dimensions; these were connected by a column-switching system composed of a 50-mL loop, three three-port valves, and a switch. 2D-CCC was used for preparativeseparation of diterpenoids (oridonin and ponicidin) from acrude extract of Rabdosia rubescens by use of solventsystems composed of n-hexane–ethyl acetate–methanol–water (1:5:1:5 and 3:5:3:5, v/v). The 2D-CCC approachwas also successfully applied to the isolation of threeprenylflavonoids from Artocarpus altilis with a pair oftwo-phase solvent systems composed of n-hexane–ethylacetate–methanol–water (5:5:7:3 and 5:5:6.5:3.5, v/v) [48].

Recently, 2D-CCC was further improved by use of a solid-phase trapping interface between the two dimensions. In thisstudy, Hu et al. [49] built a system including a semi-preparative

Fig. 5 Schematic representation of an MDCCC system. In configura-tion A, sample is injected through the six-port injection valve whilepump 2 is stopped. In configuration B, the peak of interest from

HSCCC 1 is sent to HSCCC 2 via the second six-port switching valve.Adapted from Ref. [43]


CCC instrument (Vc=140 mL) in the first dimension and aparallel three-coil CCC instrument (40 mL each coil) in thesecond dimension. By use of a solid-phase trapping columnand switching valves, the fractions of interest in the firstcolumn were concentrated and introduced on-line to the sec-ond column. The authors mentioned that up to three indepen-dent CCC separations can be performed at the same time in thesecond dimension, which, de facto, greatly increases separa-tion efficiency. This approach was used for the separation ofhydroxyanthraquinones from Rheum officinale, using elution–extrusion in the first dimension and acid–base elution in thesecond dimension. A biphasic solvent system composed of n-hexane–ethyl acetate–methanol–water (1:1:1:1, v/v) was usedto rapidly fractionate the crude extract. Two fractions were thenconcentrated online and further separated in the second dimen-sion with a solvent system composed of methyl tert-butylether–acetonitrile–water (2:2:3, v/v), with trifluoroacetic acidand triethylamine added to the upper organic phase and theaqueous lower phase, respectively, as retainer and eluter. Finalimprovement of the 2D-CCCwas achieved by development ofintegrated online column-switching CCC with a solid-phasetrapping and preconcentration interface [50]. In this case, targetcompounds were captured online on the solid-phase trappingcolumn. Eluent from the first CCC was evaporated, and themolecules were then eluted into the second CCC with thecorresponding mobile phase. This MDCCC system resultedin better orthogonality and avoided transfer of large volume ofsolvent into the subsequent CCC separation. Furthermore, bothmultilayer-coil columns were integrated into a normal centri-fuge apparatus by addition of only one more flow tube, whichkeeps columns independent of each other. Application of thissystem was demonstrated by preparative separation of antiox-idant compounds from Rubia cordifolia.

On line HPLC monitoring

Use of CCC or CPC to fractionate or purify natural productsgenerates many collection tubes whose contents must bechecked by further HPLC or thin-layer chromatography(TLC). This entire procedure is tedious, time consuming,and can lead to compound degradation. As a consequence,the last decade has seen the development of interestingseparation techniques consisting of direct hyphenation ofCCC or CPC with HPLC instruments enabling on-line mon-itoring of the preparative separation by HPLC–UV.

HSCCC-HPLC monitoring

The first application of automated HSCCC–HPLC–DADwas proposed by Zhou et al. in 2006 for isolation andpurification of hyperoside from Hypericum perforatum[51] and xanthones from Anemarrhena asphodeloides[52]. In this approach the HSCCC and HPLC instrumentswere interconnected via a T-splitter and a six-port switchvalve (Fig. 7). The T-splitter diverted 1/20 of the HSCCCflow to the switching valve. HSCCC effluent was stored inthe injection loop and, when triggered, the valve sent thefraction into the HPLC column. This HSCCC–HPLC–DADwas also successfully used for isolation of polymethoxylatedflavones from Taraxacum mongolicum [53].

Recently, Wu’s group developed a two dimensional CCC× LC system connected by a six-port valve for purificationof arctiin from Arctium lappa [54]. First the CCC dimensionwas optimized for fractionation of crude extract and isola-tion of arctiin by use of a solvent system containing salt.Macroporous resin chromatography was then used in thesecond dimension to adsorb, desalt, and desorb the arctiinfrom the CCC effluent containing NaCl. The same teamfurther developed a stop-and-go two dimensional CCC ×LC system based on their previous work [55]. The stop-and-go technique, commonly used in LC × LC, involves stop-ping elution from the first dimension while a fraction istransferred to and analysed in second dimension. Elutionin the first dimension is then resumed while the seconddimension is re-equilibrated with the mobile phase [56,57]. In this way, the authors partially resolved the timeconstraints of the second dimension, which must be rapidyet enable collection, transfer, and analysis of the fractionbefore the next one. Application of this approach led to theisolation and purification of two novel flavonoids fromMedicago sativa by use of an isopropanol–20 % NaClsolvent system (1:1, v/v). Therefore, coupling of CCC withmacroporous resin LC can be regarded as a simple tech-nique for removal of salt from CCC effluent without addi-tional steps.

A new on-line HSCCC coupled with semi-preparativeLC has recently been set up for isolation of four phthalide

Fig. 6 Multidimensional counter-current chromatograms of the agly-cone flavonoids from G. biloba. I, isorhamnetin; K, kaempferol; Q,quercetin. (A) Chromatogram obtained by HSCCC 1; (B) Chromato-gram obtained by HSCCC 2. Reprinted from Ref. [43], withpermission


compounds from Chuanxiong Rhizoma [58]. Overlappedcompounds were transferred online from HSCCC andinjected into semi-preparative LC through the six-way valvefor further separation. Transfer was possible because of thepresence of water, methanol, and acetonitrile in the HSCCCmobile phase.

CPC-HPLC monitoring

The first hyphenated CPC–HPLC system was developed byMichel et al. in 2011 [59]. This approach consists of a semi-preparative CPC–evaporative light scattering system (CPC–ELSD) in the first dimension coupled to an HPLC–UVequipped with a fused core C18 column in the second di-mension. A manual six-port valve was used to interface thetwo instruments and an additional variable flow splitter wasused to divide CPC effluent through ELSD and fractioncollector. In this case, use of a fused core column enabledrapid separation (7 min) with a good resolution in thesecond dimension, and, consequently, an increase in thenumber of possible transfers between CPC and HPLC. Theauthors also reported that CPC effluent could be directlyinjected into the C18 column irrespective of whether thesample was dissolved in the aqueous or organic CPC phaseif the injection volume did not exceed 20 μL. This method-ology was used to fractionate an extract of H. rhamnoidesberries and obtain an on-line fingerprint of collected frac-tions, which reduced post-fractionation analyticalmonitoring.

The same group recently improved this approach bycoupling CPC–HPLC to both DAD and MS detectors; thisenables simultaneous separation, purification, and identifi-cation of molecules from plant extracts [60]. Addition ofDAD and MS detectors enables double on-line purity mon-itoring with enhanced sensitivity and MS gives immediatestructural information about the separated compounds. Fur-thermore, in this work the system was fully integrated and

automated by the use of an SCPC-250 instrument linked to aSpot Prep II, with both controlled by the Armen Glider CPC(AGC) software. A diagram of the complete arrangement isshown in Fig. 8. The heart of the system is the automatic six-port switching valve enabling transfer of CPC eluent (20 μL)into the reversed-phase monolithic column of the secondHPLC dimension. When the switching valve is in the “load”position, the CPC effluent passes through the injection loopand is then sent to the CPC–UV detector and to the fractioncollector while the HPLC column is equilibrated. When atarget peak appeared the six-port valve was automaticallyturned to the “inject” position. CPC effluent present in the loopat that time is directly injected into the second HPLC–DAD–MS dimension. At the same time, the CPC system and fractioncollector continue to operate. The valve is returned to its initialposition after 2 s to ensure complete injection of the sample.This on-line strategy was well applied to the separation, puri-fication, and identification on a semi-preparative scale of xan-thones from Garcinia mangostana pericarp. α-Mangostin andγ-mangostin were both purified at a yield over 98 %, and 16other xanthones were collected in different fractions and iden-tified on the basis of their UVand mass spectra.

Off-line CCC-GC monitoring

Advances in analytical chemistry now enable on-line cou-pling of CCC and GC because of their different mobilephase nature (i.e. liquid and gas phases). However, numer-ous work has seen the development of interesting off-lineprocedures involving first CCC fractionation and then GCanalysis of fractions. For instance, Inui et al. [61] elaborateda biochemometric method consisting in four main steps:

1. preparative fractionation of crude extracts by CCC;2. biological evaluation of the fractions obtained and gen-

eration of a high-resolution biochromatogram;

Fig. 7 Schematic diagram of the hyphenated HSCCC–HPLC–DAD designed by Zhou et al. Reprinted from Ref. [51], with permission


3. GC–MS analysis of all CCC fractions and subsequentbuilding of a three-dimensional CCC–GC–MS data ma-trix; and

4. data processing and chemometric analysis enabling assign-ment of the bioactive molecules in the complex extract.

This approach was evaluated for identification of anti-tuberculosis compounds from Oplopanax horridus.Biochemometric analysis identified the 100 most activeconstituents from the crude extract by use of orthogonalCCC and GC–MS results. With help from the literature,positive correlations enabled distinction of 29 bioactivecompounds from inactive ones belonging to three mainstructural classes. Kapp and Vetter used offline coupling ofHSCCC and GC–MS for fractionation of toxaphene com-pounds [62]. Creation of a two-dimensional contour plot fromHSCCC–GC–MS results provided an excellent view of thefractionation process. Furthermore, the 2D chromatographicprocess improved the separation and identification of toxa-phene compounds compared with unidimensional GC.

On-line method for simultaneous extractionand isolation

An emerging trend in CCC coupling strategy seems to be thecombination of one modern extraction technique, for exampleaccelerated solvent extraction (ASE) or microwave-assistedextraction (MAE), with a CCC instrument, to reduce total

extraction–isolation time and to avoid degradation of unstableconstituents by light and/or oxygen. In 2011, Zhang et al. werethe first to combine the advantages of ASE extraction andCCC [63]. Figure 9 depicts the two hyphenation strategieswhich they evaluated. In the first (set up A), the ASE andHPCCC instruments are directly connected by a sample loop(100.0 cm, 0.2 mm i.d.). Plant material is extracted using thelower and/or upper phases of the CCC system and the liquidextract is then directly introduced through the HPCCC inletwith the aid of ASE pressure. In the second (set up B), theASE and HPCCC are linked via a set of two T-splitters. Thissystem is also equipped with a sample loop (400.0 cm, 2 mmi.d.) and an auxiliary pump. Both configurations have beensuccessfully assessed for extraction and on-line isolation offive molecules from Hypericum perforatum. However bothhave advantages and disadvantages: the first achieves station-ary phase retention >85% and is easy to set up, but it is limitedby the injection volume, which reduces the quantity of sampleinjected into the HPCCC column and eventually affects theyield; the second enables injection of a large volume of extractsuitable for preparative isolation, but retention of the station-ary phase is lower.

The same Chinese group recently used the secondconfiguration for the online extraction and isolation ofsaponins from Panax notoginseng [64]. In this workthey used three-stage temperature-gradient ASE coupledwith HPCCC, enabling isolation of nine saponins witha wide range of polarity in a short time. The upperphases of different solvent systems were systematically

Fig. 8 Schematic diagram ofon-line CPC–HPLC–DAD–MS. The purple arrowsrepresent CPC flow whereas thegreen arrows depict HPLCflow. (a) Six-port switchingvalve configuration in position“load”. (b) Six-port switchingvalve configuration in position“inject”. Reprinted from Ref.[60], with permission


chosen as extraction solvents and stationary phases, andthe compounds of interest were then eluted with thecorresponding lower phases.

Tong et al. set up an on-line method based onhyphenation between dynamic microwave-assisted ex-traction (DMAE) and HSCCC [65]. The authors welldemonstrated that the crude extracts obtained fromDMAE could be directly introduced into the HSCCCsystem, including a concentration step, for continuousisolation of nevadensin from Lyeicnotus pauciflorus.During MAE extraction, the sample is sent to an auto-matic concentrator which evaporates the extract at50 °C. The dry extract is then dissolved in the lowerphase of the solvent system n-hexane–ethyl acetate–methanol–water (7:3:5:5, v/v ) and transferred into thesample loop of the automatic injection valve. Whensample injection was complete, the sample was intro-duced into the HSCCC column and separated by use ofthe above solvent system. After 20 min, the valve canbe switched back to its initial position for the nextsample injection.

On-line method for simultaneous isolations

The first multi-channel countercurrent chromatographyMC–CCC apparatus was designed by Wu et al. for multi-channel fractionation of natural products [66]. It consistedof three independent coils connected by parallel flow tubesin PTFE thus forming three independent CCC columnsarranged symmetrically around the centrifuge axes. Eachcolumn was able to undergo identical synchronous planetarymotion and had a volume of 300 mL. Therefore a singleapparatus can simultaneously perform three different sepa-rations. However, this procedure requires three constant-flow pumps, three injection valves, three detectors, andthree fraction collectors (Fig. 10). An additional requirementis the use of solvent systems of similar density, to balancethe centrifuge system. The authors assessed MC–CCC per-formance by parallel high-throughput fractionation of threenaturals extracts (Solidago canadensis, Suillus placidus, andTrichosanthes kirilowii). Further combination of biologicalscreening and HPLC purification of obtained fractions led topurification of cytotoxic compounds. More recently, MC–

Fig. 9 Schematicrepresentation of the two set upsenabling on-line couplingbetween ASE and HPCCC. Setup A: the ASE and HPCCCinstruments are directlyconnected by a sample loop(100.0 cm, 0.2 mm i.d.). Set upB: the ASE and HPCCC arelinked via a set of two T-splitters plus a sample loop(400.0 cm, 2 mm i.d.).Reprinted from Ref. [63], withpermission.

Fig. 10 Schematic representation of multi-channel CCC fractionation system. Adapted from Ref. [68]


CCC has been used for fractionation of antioxidant com-pounds from Chinese medicinal plants [67, 68]. Overall,MC–CCC combined with bioactivity tests and additionalpurification techniques reduces separation time and im-proves the entire process, leading to bioactive targets.

On-line method for simultaneous isolation and activityevaluation

CCC techniques have been widely used in combination withbioactivity tests, especially for bioassay-guided fraction-ation studies [14, 69]. Indeed, after preparative separation,the activity of the collected fractions was usually assessedby use of off-line methods. This approach requires drying ofthe fractions and redissolution of the sample in an appropri-ate solvent, which is time-consuming and enhances the riskof errors. Development of an on-line method for simulta-neous fractionation and activity evaluation would avoidthese problems. Nevertheless, as far as we are aware onlyone study reports direct on-line HSCCC with bioactivitytesting. Shi et al. designed a hyphenated high-throughputpreparative isolation–activity evaluation system by couplingHSCCC on-line with radical scavenging detection(HSCCC–DPPH) [70]. After the HSCCC separation, theeffluent was split into two streams by use of an adjustablehigh-pressure stream splitter. The main portion was sentthrough the detector and the fraction collector whereas onlya small part (0.1 mL min−1) was sent to a secondary coil foron-line radical-scavenging detection. The length of the tubeused for the post-column reaction was adjusted to achieve areaction time of 0.6 min. An auxiliary pump furnishedDPPH solution at 1.1 mL min−1. The reaction responsewas finally recorded at 517 nm. This strategy was performedfor isolation of four antioxidants which were found to be themain radical scavengers from Selaginella moellendorffii.

Conclusion

Countercurrent separation, which involves partition ofanalytes between two immiscible phases, has been increas-ingly used for extraction and purification of natural prod-ucts. As described in this review, CCC has evolved duringthe last decade with the development of innovative couplingstrategies, for example hyphenation with MS and NMRdetectors, MDCCC, 2D-CCC, or on-line HPLC monitoring.On-line coupling strategies have enabled saving of time inthe entire purification process, by combining two or threecritical steps (e.g. extraction–separation, separation–identi-fication), and increasing of peak capacity, leading to a betterisolation of targeted compounds. The monitoring of CCCeffluent with MS and NMR affords direct characterization of

isolated molecules, and monitoring by HPLC enables an on-line purity monitoring of target compounds.

In France, research groups have been involved in thesedevelopments and have contributed to the last CPC evolu-tion in collaboration with the two worldwide French CPCmanufacturers (i.e. Armen instrument and Kromaton groupRousselet–Robatel).

Although significant progress has been made in CCC,many aspects related to coupling feasibility, detection sen-sitivity, and system automation require additional scientificeffort to enable routine use of on-line coupling of CCCstrategies in any laboratory. Future CCC evolution will tendtoward the development of more integrated systems, forexample CPC–HPLC–DAD–MS combining separation,analysis, and identification in an unique single step. Anotherinteresting prospect is on-line hyphenation of CCC withbioactivity tests, enabling the development of high-throughput screening and saving of time in the discoveryof bioactive compounds.

Finally, we hope this review will attract analytical andnatural product chemists to work on HSCCC and CPC de-velopments and also challenge other scientists outside thearea of CCC to share efforts, resulting in more interdisci-plinary concepts.

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