Analytical and Bioanalytical Chemistry

, Volume 406, Issue 4, pp 957–969 | Cite as

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.


HSCCC CPC MS On-line and off-line coupling Extraction Purification Natural products 


  1. 1.
    Berthod A, Maryutina T, Spivakov B, Shpigun O, Sutherland IA (2009) Countercurrent chromatography in analytical chemistry. Pure Appl Chem 81:355–387CrossRefGoogle Scholar
  2. 2.
    Ito Y (2005) Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J Chromatogr A 1065:145–168CrossRefGoogle Scholar
  3. 3.
    Marchal L, Legrand J, Foucault A (2003) Centrifugal partition chromatography: a survey of its history, and our recent advances in the field. Chem Rec 3:133–143CrossRefGoogle Scholar
  4. 4.
    Marston A, Hostettmann K (2006) Developments in the application of counter-current chromatography to plant analysis. J Chromatogr A 1112:181–194CrossRefGoogle Scholar
  5. 5.
    DeAmicis C, Edwards NA, Giles MB, Harris GH, Hewitson P, Janaway L, Ignatova S (2011) Comparison of preparative reversed phase liquid chromatography and countercurrent chromatography for the kilogram scale purification of crude spinetoram insecticide. J Chromatogr A 1218:6122–6127CrossRefGoogle Scholar
  6. 6.
    Pauli GF, Pro SM, Friesen JB (2008) Countercurrent separation of natural products. J Nat Prod 71:1489–1508CrossRefGoogle Scholar
  7. 7.
    Winterhalter P (2007) Application of countercurrent chromatography (CCC) to the analysis of natural pigments. Trends Food Sci Technol 18:507–513CrossRefGoogle Scholar
  8. 8.
    Yoon KD, Chin Y-W, Kim J (2010) Centrifugal partition chromatography: application to natural products in 1994–2009. J Liq Chromatogr Relat Technol 33:1208–1254CrossRefGoogle Scholar
  9. 9.
    Hu R, Pan Y (2012) Recent trends in counter-current chromatography. Trends Anal Chem 40:15–27CrossRefGoogle Scholar
  10. 10.
    Herrero M, Ibáñez E, Cifuentes A, Bernal J (2009) Multidimensional chromatography in food analysis. J Chromatogr A 1216:7110–7129CrossRefGoogle Scholar
  11. 11.
    Johnson TA, Sohn J, Inman WD, Estee SA, Loveridge ST, Vervoort HC, Tenney K, Liu J, Ang KK-H, Ratnam J, Bray WM, Gassner NC, Shen YY, Lokey RS, McKerrow JH, Boundy-Mills K, Nukanto A, Kanti A, Julistiono H, Kardono LBS, Bjeldanes LF, Crews P (2011) Natural product libraries to accelerate the high-throughput discovery of therapeutic leads. J Nat Prod 74:2545–2555CrossRefGoogle Scholar
  12. 12.
    Palma P, Famiglini G, Trufelli H, Pierini E, Termopoli V, Cappiello A (2011) Electron ionization in LC–MS: recent developments and applications of the direct-EI LC–MS interface. Anal Bioanal Chem 399:2683–2693CrossRefGoogle Scholar
  13. 13.
    Zehl M, Braunberger C, Conrad J, Crnogorac M, Krasteva S, Vogler B, Beifuss U, Krenn L (2011) Identification and quantification of flavonoids and ellagic acid derivatives in therapeutically important Drosera species by LC–DAD, LC–NMR, NMR, and LC–MS. Anal Bioanal Chem 400:2565–2576CrossRefGoogle Scholar
  14. 14.
    Abdellaoui S, Destandau E, Toribio A, Elfakir C, Lafosse M, Renimel I, André P, Cancellieri P, Landemarre LS (2010) Bioactive molecules in Kalanchoe pinnata leaves: extraction, purification, and identification. Anal Bioanal Chem 398:1329–1338CrossRefGoogle Scholar
  15. 15.
    Cicek SS, Schwaiger S, Ellmerer EP, Stuppner H (2010) Development of a fast and convenient method for the isolation of triterpene saponins from Actaea racemosa by high-speed countercurrent chromatography coupled with evaporative light scattering detection. Planta Med 76:467–473CrossRefGoogle Scholar
  16. 16.
    Michel T, Destandau E, Pecher V, Renimel I, Pasquier L, André P, Elfakir C (2011) Two-step Centrifugal Partition Chromatography (CPC) fractionation of Butea monosperma (Lam.) biomarkers. Sep Purif Technol 80:32–37CrossRefGoogle Scholar
  17. 17.
    Yoon KD, Chin Y-W, Yang MH, Choi J, Kim J (2012) Application of high-speed countercurrent chromatography–evaporative light scattering detection for the separation of seven steroidal saponins from Dioscorea villosa. Phytochem Anal 23:462–468CrossRefGoogle Scholar
  18. 18.
    Lee YW, Voyksner RD, Fang QC, Cook CE, Ito Y (1988) Application of countercurrent chromatography/thermospray mass spectrometry for the analysis of natural products. J Liq Chromatogr 11:153–171CrossRefGoogle Scholar
  19. 19.
    Lee YW, Voyksner RD, Pack TW, Cook CE, Fang QC, Ito Y (1990) Application of countercurrent chromatography/thermospray mass spectrometry for the identification of bioactive lignans from plant natural products. Anal Chem 62:244–248CrossRefGoogle Scholar
  20. 20.
    Oka H, Ikai Y, Kawamura N, Hayakawa J, Harada K, Murata H, Suzuki M, Ito Y (1991) Direct interfacing of high-speed countercurrent chromatography to frit electron ionization, chemical ionization, and fast atom bombardment mass spectrometry. Anal Chem 63:2861–2865CrossRefGoogle Scholar
  21. 21.
    McGuire James N, Proefke Mark L, Conway Walter D, Rinehart Kenneth L (1995) On-line fast atom bombardment mass spectrometric detection in high-speed countercurrent chromatography through a moving belt interface. In: Modern Countercurrent Chromatography, vol 593. ACS Symposium Series, vol 593. American Chemical Society, pp 129–142Google Scholar
  22. 22.
    Ito Y, Goto T, Yamada S, Ohno T, Matsumoto H, Oka H, Ito Y (2008) Rapid determination of carbamate pesticides in food using dual counter-current chromatography directly interfaced with mass spectrometry. J Chromatogr A 1187:53–57CrossRefGoogle Scholar
  23. 23.
    Kidwell H, Jones JJ, Games DE (2001) Separation and characterisation of five polar herbicides using countercurrent chromatography with detection by negative ion electrospray ionisation mass spectrometry. Rapid Commun Mass Spectrom 15:1181–1186CrossRefGoogle Scholar
  24. 24.
    Inoue K, Hattori Y, Hino T, Oka H (2010) An approach to on-line electrospray mass spectrometric detection of polypeptide antibiotics of enramycin for high-speed counter-current chromatographic separation. J Pharm Biomed Anal 51:1154–1160CrossRefGoogle Scholar
  25. 25.
    Inoue K, Hattori Y, Hino T, Oka H (2011) Preparative isolation of avermectin isomers by high-speed countercurrent chromatography coupled with electrospray mass spectrometry. J Liq Chromatogr Relat Technol 34:2621–2628CrossRefGoogle Scholar
  26. 26.
    Inoue K, Hattori Y, Horie M, Tomoaki H, Oka H (2011) Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry. J Sep Sci 34:1484–1488CrossRefGoogle Scholar
  27. 27.
    Jones JJ, Kidwell H, Games DE (2003) Application of atmospheric pressure chemical ionisation mass spectrometry in the analysis of barbiturates by high-speed analytical countercurrent chromatography. Rapid Commun Mass Spectrom 17:1565–1572CrossRefGoogle Scholar
  28. 28.
    Kong Z, Rinehart KL, Milberg RM, Conway WD (1998) Application of high-speed countercurrent chromatography/electrospray ionization mass spectrometry (HSCCC/ESIMS) in natural products chemistry. J Liq Chromatogr Relat Technol 21:65–82CrossRefGoogle Scholar
  29. 29.
    Chen LJ, Song H, Du QZ, Li JR, Ito Y (2005) Analysis of flavonoids in the extracts from the seeds of Oroxylum indicum using high speed countercurrent chromatography/mass spectrometry. J Liq Chromatogr Relat Technol 28:1549–1555CrossRefGoogle Scholar
  30. 30.
    Gutzeit D, Winterhalter P, Jerz G (2007) Application of preparative high-speed counter-current chromatography/electrospray ionization mass spectrometry for a fast screening and fractionation of polyphenols. J Chromatogr A 1172:40–46CrossRefGoogle Scholar
  31. 31.
    Hou Z, Xu D, Yao S, Luo J, Kong L (2009) An application of high-speed counter-current chromatography coupled with electrospray ionization mass spectrometry for separation and online identification of coumarins from Peucedanum praeruptorum Dunn. J Chromatogr B 877:2571–2578CrossRefGoogle Scholar
  32. 32.
    Jerz G, Murillo-Velásquez JA, Skrjabin I, Gök R, Winterhalter P (2012) Anacardic acid profiling in cashew nuts by direct coupling of preparative high-speed countercurrent chromatography and mass spectrometry (prepHSCCC–ESI–/APCI–MS–MS). In: Recent Advances in the Analysis of Food and Flavors, vol 1098. ACS Symposium Series, vol 1098. American Chemical Society, pp 145–165Google Scholar
  33. 33.
    Jerz G, Gutzeit D, Winterhalter P (2010) Characterization of acylated flavonoid glycosides from sea buckthorn (Hippophaë rhamnoides) juice concentrate by preparative HSCCC/ESI–MS–MS. In: Flavor and Health Benefits of Small Fruits, vol 1035. ACS Symposium Series, vol 1035. American Chemical Society, pp 253–265Google Scholar
  34. 34.
    Jerz G, Wybraniec S, Gebers N, Winterhalter P (2010) Target-guided separation of Bougainvillea glabra betacyanins by direct coupling of preparative ion-pair high-speed countercurrent chromatography and electrospray ionization mass-spectrometry. J Chromatogr A 1217:4544–4554CrossRefGoogle Scholar
  35. 35.
    Destandau E, Toribio A, Lafosse M, Pecher V, Lamy C, André P (2009) Centrifugal partition chromatography directly interfaced with mass spectrometry for the fast screening and fractionation of major xanthones in Garcinia mangostana. J Chromatogr A 1216:1390–1394CrossRefGoogle Scholar
  36. 36.
    Toribio A, Destandau E, Elfakir C, Lafosse M (2009) Hyphenation of centrifugal partition chromatography with electrospray ionization mass spectrometry using an active flow-splitter device for characterization of flavonol glycosides. Rapid Commun Mass Spectrom 23:1863–1870CrossRefGoogle Scholar
  37. 37.
    Bisson J, Poupard P, Pawlus AD, Pons A, Darriet P, Mérillon J-M, Waffo-Téguo P (2011) Development of hybrid elution systems for efficient purification of stilbenoids using centrifugal partition chromatography coupled to mass spectrometry. J Chromatogr A 1218:6079–6084CrossRefGoogle Scholar
  38. 38.
    Janaway L, Hawes D, Ignatova S, Wood P, Sutherland IA (2003) A new small coil-volume CCC instrument for direct interfacing with MS. J Liq Chromatogr Relat Technol 26:1345–1354CrossRefGoogle Scholar
  39. 39.
    Spraul M, Braumann U, Renault J-H, Thépenier P, Nuzillard J-M (1997) Nuclear magnetic resonance monitoring of centrifugal partition chromatography in pH-zone-refining mode. J Chromatogr A 766:255–260CrossRefGoogle Scholar
  40. 40.
    Bisson J, Brunel M, Badoc A, Palos-Pinto A, Merillon JM, Waffo-Teguo P (2012) Hyphenating countercurrent chromatography with NMR and mass spectrometry. How to enhance the range of the liquid phases. Planta Med 78:CL48CrossRefGoogle Scholar
  41. 41.
    Marchal A, Waffo-Téguo P, Génin E, Mérillon J-M, Dubourdieu D (2011) Identification of new natural sweet compounds in wine using centrifugal partition chromatography–gustatometry and Fourier transform mass spectrometry. Anal Chem 83:9629–9637CrossRefGoogle Scholar
  42. 42.
    Evans C, Jorgenson J (2004) Multidimensional LC–LC and LC–CE for high-resolution separations of biological molecules. Anal Bioanal Chem 378:1952–1961CrossRefGoogle Scholar
  43. 43.
    Yang F, Quan J, Zhang TY, Ito Y (1998) Multidimensional counter-current chromatographic system and its application. J Chromatogr A 803:298–301CrossRefGoogle Scholar
  44. 44.
    Wei Y, Ito Y (2006) Preparative isolation of imperatorin, oxypeucedanin and isoimperatorin from traditional Chinese herb "bai zhi" Angelica dahurica (Fisch. ex Hoffm) Benth. et Hook using multidimensional high-speed counter-current chromatography. J Chromatogr A 1115:112–117CrossRefGoogle Scholar
  45. 45.
    Wei J, Zhang T, Ito Y (2005) Preparative separation of tripdiolide from Chinese traditional herb by multidimensional CCC. J Liq Chromatogr Relat Technol 28:1903–1911CrossRefGoogle Scholar
  46. 46.
    Tian G, Zhang T, Zhang Y, Ito Y (2002) Separation of tanshinones from Salvia miltiorrhiza Bunge by multidimensional counter-current chromatography. J Chromatogr A 945:281–285CrossRefGoogle Scholar
  47. 47.
    Lu Y, Sun C, Liu R, Pan Y (2007) Effective two-dimensional counter-current chromatographic method for simultaneous isolation and purification of oridonin and ponicidin from the crude extract of Rabdosia rubescens. J Chromatogr A 1146:125–130CrossRefGoogle Scholar
  48. 48.
    Lu Y, Sun C, Wang Y, Pan Y (2007) Two-dimensional counter-current chromatography for the preparative separation of prenylflavonoids from Artocarpus altilis. J Chromatogr A 1151:31–36CrossRefGoogle Scholar
  49. 49.
    Hu R, Dai X, Xu X, Sun C, Pan Y (2011) Two-dimensional counter-current chromatography: 1st Traditional counter-current chromatography, 2nd acid–base elution counter-current chromatography. J Chromatogr A 1218:6085–6091CrossRefGoogle Scholar
  50. 50.
    Lu Y, Hu R, Pan Y (2010) Integrated countercurrent extraction of natural products: a combination of liquid and solid supports. Anal Chem 82:3081–3085CrossRefGoogle Scholar
  51. 51.
    Zhou T, Chen B, Fan G, Chai Y, Wu Y (2006) Application of high-speed counter-current chromatography coupled with high-performance liquid chromatography–diode array detection for the preparative isolation and purification of hyperoside from Hypericum perforatum with online purity monitoring. J Chromatogr A 1116:97–101CrossRefGoogle Scholar
  52. 52.
    Zhou T, Zhu Z, Wang C, Fan G, Peng J, Chai Y, Wu Y (2007) On-line purity monitoring in high-speed counter-current chromatography: Application of HSCCC–HPLC–DAD for the preparation of 5-HMF, neomangiferin and mangiferin from Anemarrhena asphodeloides Bunge. J Pharm Biomed Anal 44:96–100CrossRefGoogle Scholar
  53. 53.
    Shi S, Zhou H, Zhang Y, Zhao Y, Huang K, Liu S (2009) A high-speed counter-current chromatography–HPLC–DAD method for preparative isolation and purification of two polymethoxylated flavones from Taraxacum mongolicum. J Chromatogr Sci 47:349–353CrossRefGoogle Scholar
  54. 54.
    Guo M, Liang J, Wu S (2010) On-line coupling of counter-current chromatography and macroporous resin chromatography for continuous isolation of arctiin from the fruit of Arctium lappa L. J Chromatogr A 1217:5398–5406CrossRefGoogle Scholar
  55. 55.
    Liang J, Yang Z, Cao X, Wu B, Wu S (2011) Preparative isolation of novel antioxidant flavonoids of alfalfa by stop-and-go counter-current chromatography and following on-line liquid chromatography desalination. J Chromatogr A 1218:6191–6199CrossRefGoogle Scholar
  56. 56.
    Fairchild JN, Horváth K, Guiochon G (2009) Approaches to comprehensive multidimensional liquid chromatography systems. J Chromatogr A 1216:1363–1371CrossRefGoogle Scholar
  57. 57.
    Guiochon G, Marchetti N, Mriziq K, Shalliker RA (2008) Implementations of two-dimensional liquid chromatography. J Chromatogr A 1189:109–168CrossRefGoogle Scholar
  58. 58.
    Wei Y, Huang W, Gu Y (2013) Online isolation and purification of four phthalide compounds from Chuanxiong rhizoma using high-speed counter-current chromatography coupled with semi-preparative liquid chromatography. J Chromatogr A 1284:53–58CrossRefGoogle Scholar
  59. 59.
    Michel T, Destandau E, Elfakir C (2011) On-line hyphenation of Centrifugal Partition Chromatography (CPC) and High Pressure Liquid Chromatography (HPLC) for the fractionation of flavonoids from Hippophaë rhamnoides L. berries. J Chromatogr A 1218:6173–6178CrossRefGoogle Scholar
  60. 60.
    Michel T, Destandau E, Fougère L, Elfakir C (2012) New “hyphenated” CPC–HPLC–DAD–MS strategy for simultaneous isolation, analysis and identification of phytochemicals: application to xanthones from Garcinia mangostana. Anal Bioanal Chem 404:2963–2972CrossRefGoogle Scholar
  61. 61.
    Inui T, Wang Y, Pro SM, Franzblau SG, Pauli GF (2012) Unbiased evaluation of bioactive secondary metabolites in complex matrices. Fitoterapia 83:1218–1225CrossRefGoogle Scholar
  62. 62.
    Kapp T, Vetter W (2009) Offline coupling of high-speed counter-current chromatography and gas chromatography/mass spectrometry generates a two-dimensional plot of toxaphene components. J Chromatogr A 1216:8391–8397CrossRefGoogle Scholar
  63. 63.
    Zhang Y, Liu C, Yu M, Zhang Z, Qi Y, Wang J, Wu G, Li S, Yu J, Hu Y (2011) Application of accelerated solvent extraction coupled with high-performance counter-current chromatography to extraction and online isolation of chemical constituents from Hypericum perforatum L. J Chromatogr A 1218:2827–2834CrossRefGoogle Scholar
  64. 64.
    Zhang Y, Liu C, Qi Y, Li S, Wang J (2013) Application of accelerated solvent extraction coupled with counter-current chromatography to extraction and online isolation of saponins with a broad range of polarity from Panax notoginseng. Sep Purif Technol 106:82–89CrossRefGoogle Scholar
  65. 65.
    Tong X, Xiao X, Li G (2011) On-line coupling of dynamic microwave-assisted extraction with high-speed counter-current chromatography for continuous isolation of nevadensin from Lyeicnotus pauciflorus Maxim. J Chromatogr B 879:2397–2402CrossRefGoogle Scholar
  66. 66.
    Wu S, Yang L, Gao Y, Liu X, Liu F (2008) Multi-channel counter-current chromatography for high-throughput fractionation of natural products for drug discovery. J Chromatogr A 1180:99–107CrossRefGoogle Scholar
  67. 67.
    Hu R, Lu Y, Dai X, Pan Y (2010) Screening of antioxidant phenolic compounds in Chinese Rhubarb combining fast counter-current chromatography fractionation and liquid chromatography/mass spectrometry analysis. J Sep Sci 33:1595–1603CrossRefGoogle Scholar
  68. 68.
    Lu Y, Berthod A, Hu R, Ma W, Pan Y (2009) Screening of complex natural extracts by countercurrent chromatography using a parallel protocol. Anal Chem 81:4048–4059CrossRefGoogle Scholar
  69. 69.
    Shi S-Y, Peng M-J, Zhang Y-P, Peng S (2013) Combination of preparative HPLC and HSCCC methods to separate phosphodiesterase inhibitors from Eucommia ulmoides bark guided by ultrafiltration-based ligand screening. Anal Bioanal Chem 405(12):4213–4223Google Scholar
  70. 70.
    Shi S, Zhou H, Zhang Y, Huang K (2008) Hyphenated HSCCC–DPPH· for rapid preparative isolation and screening of antioxidants from Selaginella moellendorffii. Chromatographia 68:173–178CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Thomas Michel
    • 1
  • Emilie Destandau
    • 1
  • Claire Elfakir
    • 1
  1. 1.Université d’Orléans‐CNRSInstitut de Chimie Organique et Analytique UMR 7311Orléans cedex 2France

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