Separation of Natural Products by Countercurrent Chromatography

  • James B. McAlpine
  • J. Brent Friesen
  • Guido F. Pauli
Part of the Methods in Molecular Biology book series (MIMB, volume 864)


Countercurrent Chromatography (CCC) provides the natural product chemist with a high-resolution separatory method, which is uniquely applicable to sensitive (unstable) compounds and which allows virtually quantitative recovery of the load sample. Different instruments use different means of retaining a stationary liquid phase. The solvent system (SS) can be chosen to optimize the separatory power and the number of systems available is limitless. Several examples are provided to illustrate the power of the method and to guide the chemist in choice of an appropriate SS.

Key words

High Speed Countercurrent Chromatography (HSCCC) Centrifugal partition chromato-graphy Ito’s coil planet centrifuge pH zone refining GUESS method Two-phase solvent systems 


  1. 1.
    Craig LC, Craig D (1956) In: Weissenberger A (ed) Techniques in organic chemistry: separation and purification. Interscience Publishers, New York, pp 247–254Google Scholar
  2. 2.
    Dini I (2011) Flavonoid glycosides from Pouteria obovata (R. Br.) fruit flour. Food Chem 124:884–888CrossRefGoogle Scholar
  3. 3.
    Hostettmann K, Marston A (1990) Liquid-liquid partition chromatography in natural product isolation. Anal Chim Acta 236:63–76CrossRefGoogle Scholar
  4. 4.
    Marston A, Hostettmann K (2006) Develop-ments in the application of counter-current chromatography to plant analysis. J Chromatogr A 1112:181–194PubMedCrossRefGoogle Scholar
  5. 5.
    Ito Y (2005) Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J Chromatogr A 1065:145–168PubMedCrossRefGoogle Scholar
  6. 6.
    Sticher O (2008) Natural product isolation. Nat Prod Rep 25:517–554PubMedCrossRefGoogle Scholar
  7. 7.
    Sutherland IA, Fisher D (2009) Role of counter-current chromatography in the modernisation of Chinese herbal medicines. J Chromatogr A 1216:740–753PubMedCrossRefGoogle Scholar
  8. 8.
    Pan Y, Lu Y (2007) Recent progress in countercurrent chromatography. J Liq Chromatogr Relat Technol 30:649–679CrossRefGoogle Scholar
  9. 9.
    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
  10. 10.
    Pauli GF, Pro SM, Friesen JB (2008) Counter­current separation of natural products. J Nat Prod 71:1489–1508PubMedCrossRefGoogle Scholar
  11. 11.
    Ito Y (1986) High-speed countercurrent chromatography. CRC Crit Rev Anal Chem 17:65–143CrossRefGoogle Scholar
  12. 12.
    Ito Y, Conway WD (1984) Analytical chemistry – applied spectroscopy section, abstract 472. In: Pittsburgh Conference and Exposition, Atlantic CityGoogle Scholar
  13. 13.
    Ignatova S, Hawes D, van den Heuvel R, Hewitson P, Sutherland IA (2010) A new non-synchronous preparative counter-current centrifuge – the next generation of dynamic extraction/chromatography devices with independent mixing and settling control, which offer a step change in efficiency. J Chromatogr A 1217:34–39PubMedCrossRefGoogle Scholar
  14. 14.
    Sutherland IA, Hewitson P, Ignatova S (2009) Scale-up of counter-current chromatography: demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale. J Chromatogr A 1216:8787–8792PubMedCrossRefGoogle Scholar
  15. 15.
    Sutherland I, Hewitson P, Ignatova S (2009) New 18-l process-scale counter-current chromatography centrifuge. J Chromatogr A 1216:4201–4205PubMedCrossRefGoogle Scholar
  16. 16.
    Zhao Y, Du Q (2007) Separation of solanesol in tobacco leaves extract by slow rotary counter-current chromatography using a novel non-aqueous two-phase solvent system. J Chromatogr A 1151:183–196CrossRefGoogle Scholar
  17. 17.
    Du Q, Shu A, Ito Y (1996) Purification of fish oil ethyl esters by high-speed countercurrent chromatography using non-aqueous solvent systems. J Liq Chromatogr Relat Technol 19:1451–1457CrossRefGoogle Scholar
  18. 18.
    Sutherland IA, Audo G, Bourton E, Coulliard F, Fisher D, Garrard I et al (2008) Rapid linear scale-up of a protein separation by centrifugal partition chromatography. J Chromatogr A 1190:57–62PubMedCrossRefGoogle Scholar
  19. 19.
    Ruiz-Angel MJ, Pino V, Carda-Broch S, Berthod A (2007) Solvent systems for countercurrent chromatography: an aqueous two phase liquid system based on a room temperature ionic liquid. J Chromatogr A 1151:65–73PubMedCrossRefGoogle Scholar
  20. 20.
    Magri ML, Cabrera RB, Miranda MV, Fernandez-Lahore HM, Cascone O (2003) Performance of an aqueous two-phase-based countercurrent chromatographic system for horseradish peroxidase purification. J Sep Sci 26:1701–1706CrossRefGoogle Scholar
  21. 21.
    Friesen JB, Pauli GF (2005) G.U.E.S.S. – a generally useful estimations of solvent systems in CCC. J Liq Chromatogr Relat Technol 28:2777–2806CrossRefGoogle Scholar
  22. 22.
    Friesen JB, Pauli GF (2007) Rational development of solvent system families in coun-tercurrent chromatography. J Chromatogr A 1151:51–59PubMedCrossRefGoogle Scholar
  23. 23.
    Guzlek H, Wood PL, Janaway L (2009) Performance comparison using the GUESS mixture to evaluate counter-current chromatography instruments. J Chromatogr A 1216:4181–4186PubMedCrossRefGoogle Scholar
  24. 24.
    Hewitson P, Ignatova S, Ye H, Chen L, Sutherland I (2009) Intermittent counter-current extraction as an alternative approach to purification of Chinese herbal medicine. J Chromatogr A 1216:4187–4192PubMedCrossRefGoogle Scholar
  25. 25.
    Berthod A, Ignatova S, Sutherland IA (2009) Advantages of a small-volume counter-current chromatography column. J Chromatogr A 1216:4169–4175PubMedCrossRefGoogle Scholar
  26. 26.
    Li J, Ma X, Li F, Wang J, Chen H, Wang G et al (2010) Preparative separation and purification of bufadienolides from Chinese traditional medicine of ChanSu using high-speed counter-current chromatography. J Sep Sci 33:1325–1330PubMedGoogle Scholar
  27. 27.
    Zhang Y, Liu C, Zhang Z, Qi Y, Wu G, Li S (2010) Solvent gradient elution for comprehensive separation of constituents with wide range of polarity in Apocynum venetum leaves by high-speed counter-current chromatography. J Sep Sci 33:2743–2748PubMedCrossRefGoogle Scholar
  28. 28.
    Hopmann E, Arlt W, Minceva M (2011) Solvent system selection in counter-current chromatography using conductor-like screening model for real solvents. J Chromatogr A 1218:242–250PubMedCrossRefGoogle Scholar
  29. 29.
    Klamt A (1995) Conductor-like screening model for real solvents: a new approach to the quantitative calculation of solvation phenomena. J Phys Chem 99:2224–2235CrossRefGoogle Scholar
  30. 30.
    Berthod A, Hassoun M, Ruiz-Angel MJ (2006) Liquid phase chemical compositions of the Arizona biphasic liquid system, P-17. In: INCCC (ed) CCC 2006. INCCC, Bethesda, MDGoogle Scholar
  31. 31.
    Friesen JB, Pauli GF (2008) Performance characteristics of countercurrent separation in analysis of natural products of agricultural significance. J Agric Food Chem 56:19–28PubMedCrossRefGoogle Scholar
  32. 32.
    Han Q-B, Wong L, Yang N-Y, Song J-Z, Qiao C-F, Yiu H et al (2008) A simple method to optimize the HSCCC two-phase solvent system by predicting the partition coefficient for target compound. J Sep Sci 31:1189–1194PubMedCrossRefGoogle Scholar
  33. 33.
    Dubant S, Mathews B, Higginson P, Crook R, Snowden M, Mitchell J (2008) Practical solvent system selection for counter-current separation of pharmaceutical compounds. J Chromatogr A 1207:190–192PubMedCrossRefGoogle Scholar
  34. 34.
    Guzlek H, Baptista IIR, Wood PL, Livingston A (2010) A novel approach to modelling counter-current chromatography. J Chromatogr A 1217:6230–6240PubMedCrossRefGoogle Scholar
  35. 35.
    Martin AJP, Synge RLM (1941) A new form of chromatogram employing two liquid phases. I. A theory of chromatography. II. Application to the microdetermination of the higher monoamino acids in proteins. Biochem J 35:1358–1368PubMedGoogle Scholar
  36. 36.
    de Folter J, Sutherland IA (2009) Universal counter-current chromatography modelling based on counter-current distribution. J Chromatogr A 1216:4218–4224PubMedCrossRefGoogle Scholar
  37. 37.
    Booth AJ, Sutherland IA, Lye GJ (2003) Modeling the performance of pilot-scale countercurrent chromatography: scale-up predictions and experimental verification of erythromycin separation. Biotechnol Bioeng 81:640–649PubMedCrossRefGoogle Scholar
  38. 38.
    Sutherland IA, de Folter J, Wood P (2003) Modelling CCC using an eluting countercurrent distribution model. J Liq Chromatogr Relat Technol 26:1449–1474CrossRefGoogle Scholar
  39. 39.
    Gallagher B, Friesen JB, Pauli GF (2010) Development of prEEdiCCCt – a software tool for the modeling of countercurrent separations (P-43). In: Berthod A (ed) CCC 2010. INCCC, Lyon (France)Google Scholar
  40. 40.
    Friesen JB, Pauli GF (2007) Reciprocal symmetry plots as a representation of countercurrent chromatograms. Anal Chem 79:2320–2324PubMedCrossRefGoogle Scholar
  41. 41.
    Ito Y (1996) pH-peak-focusing and pH-zone-refining countercurrent chromatography. In: Ito Y, Conway WD (eds) High-speed countercurrent chromatography. Wiley, New York, pp 121–175Google Scholar
  42. 42.
    Ito Y, Ma Y (1996) pH-zone-refining countercurrent chromatography. J Chromatogr A 753:1–36PubMedCrossRefGoogle Scholar
  43. 43.
    Wu S, Sun C, Cao X, Zhou H, Hong Z, Pan Y (2004) Preparative counter-current chromatography isolation of liensinine and its analogues from embryo of the seed of Nelumbo nucifera Gaertn. using upright coil planet centrifuge with four multilayer coils connected in series. J Chromatogr A 1041:153–162PubMedCrossRefGoogle Scholar
  44. 44.
    Duanmu Q, Li A, Sun A, Liu R, Li X (2010) Semi-preparative high-speed counter-current chromatography separation of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn by pH-gradient elution. J Sep Sci 33:1746–1751PubMedCrossRefGoogle Scholar
  45. 45.
    Zheng Z, Minglin W, Daijie W, Wenjuan D, Xiao W, Chengchao Z (2010) Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining countercurrent chromatography. J Chromatogr B 878:1647–1651CrossRefGoogle Scholar
  46. 46.
    Wang X, Geng Y, Wang D, Shi X, Liu J (2008) Separation and purification of harmine and harmaline from Peganum harmala using pH-zone-refining counter-current chromatography. J Sep Sci 31:3543–3547PubMedCrossRefGoogle Scholar
  47. 47.
    Friesen JB, Pauli GF (2009) Binary concepts and standardization in countercurrent separation technology. J Chromatogr A 1216:4237–4244PubMedCrossRefGoogle Scholar
  48. 48.
    Shen C-W, Yu T (2009) Peak shape and dispersion behavior of solutes in counter-current chromatography with a single phase. J Chromatogr A 1216:6789–6795PubMedCrossRefGoogle Scholar
  49. 49.
    Berthod A, Friesen JB, Inui T, Pauli GF (2007) Elution-extrusion countercurrent chromatography: theory and concepts in metabolic analysis. Anal Chem 79:3371–3382PubMedCrossRefGoogle Scholar
  50. 50.
    Friesen JB, Pauli GF (2009) GUESS mix-guided optimization of elution-extrusion counter-current separations. J Chromatogr A 1216:4225–4231PubMedCrossRefGoogle Scholar
  51. 51.
    Schaufelberger DE, McCloud TG, Beutler JA (1991) Laser-light-scattering detection for high-speed counter-current chromatography. J Chromatogr 538:87–90PubMedCrossRefGoogle Scholar
  52. 52.
    Han QB, Zhou Y, Feng C, Xu G, Huang S-X, Li S-L et al (2009) Bioassay guided discovery of apoptosis inducers from gamboge by high-speed counter-current chromatography and high-pressure liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry. J Chromatogr B 877:401–407CrossRefGoogle Scholar
  53. 53.
    Jerz G, Wybraniec S, Gebers N, Winterhalter P (2010) Target-guided separation of Bougain-villea glabra betacyanins by direct coupling of preparative ion-pair high-speed countercurrent chromatography and electrospray ionization mass-spectrometry. J Chromatogr A 1217:4544–4554PubMedCrossRefGoogle Scholar
  54. 54.
    Lee YS, Kim SH, Kim JK, Shin H-K, Kang Y-H, Park JHY et al (2010) Rapid identification and preparative isolation of antioxidant components in licorice. J Sep Sci 33:664–671PubMedCrossRefGoogle Scholar
  55. 55.
    Drogue S, Rolet-Menet M-C, Thiebaut D, Rosset R (1992) Separation of pristinamycins by high-speed countercurrent chromatography. I. Selection of solvent system and preliminary preparative studies. J Chromatogr 593:363–371CrossRefGoogle Scholar
  56. 56.
    Chiou FY, Kan P, Chu I-M, Lee C-J (1997) Separation of taxol and cephalomannine by countercurrent chromatography. J Liq Chromatogr Relat Technol 20:57–61CrossRefGoogle Scholar
  57. 57.
    Gunawardana G, Premachandran U, Burres NS, Whittern DN, Henry R, Spanton S et al (1992) Isolation of 9-Dihydro-13-acetylbaccatin III from Taxus canadensis. J Nat Prod 55:1686–1689CrossRefGoogle Scholar
  58. 58.
    Chen RH, Hochlowski JE, McAlpine JB, Rasmussen RR (1988) Separation and purification of macrolides using the Ito Multi-Layer Horizontal Coil Planet Centrifuge. J Liq Chromatogr 11:191–201CrossRefGoogle Scholar
  59. 59.
    De Souza PA, Rangel LP, Oigman SS, Elias MM, Ferreira-Pereira A, De Lucas NC et al (2010) Isolation of two bioactive diterpenic acids from Copaifera glycycarpa oleoresin by high-speed counter-current chromatography. Phytochem Anal 21:539–543PubMedCrossRefGoogle Scholar
  60. 60.
    Wu D, Jiang X, Wu S (2010) Direct purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography without presaturation of the two-phase solvent mixture. J Sep Sci 33:67–73PubMedCrossRefGoogle Scholar
  61. 61.
    Urbain A, Corbeiller P, Aligiannis N, Halabalaki M, Skaltsounis A-L (2010) Hydrostatic countercurrent chromatography and ultra high pressure LC: Two fast complementary separation methods for the preparative isolation and the analysis of the fragrant massoia lactones. J Sep Sci 33:1198–1203PubMedGoogle Scholar
  62. 62.
    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 Pharmaceut Biomed Anal 51:1154–1160CrossRefGoogle Scholar
  63. 63.
    Brill GM, McAlpine JB, Hochlowski JE (1985) Use of coil planet centrifuge in the isolation of antibiotics. J Liq Chromatogr 8:2259–2280CrossRefGoogle Scholar
  64. 64.
    Hochlowski JE, Brill GM, Andres WW, Spanton SG, McAlpine JB (1987) Arizonins, a new complex of antibiotics related to kalafungin. II. Isolation and characterization. J Antibiot 40:401–407PubMedGoogle Scholar
  65. 65.
    Martin DG, Biles C, Peltonen RE (1986) Countercurrent chromatography in the fractionation of natural products. Am Lab 18:21–28Google Scholar
  66. 66.
    Dawson MJ, Farthing JE, Marshall PS, Middleton RF, O’Neill MJ, Shuttleworth A et al (1992) The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity. J Antibiot 45:639–647PubMedGoogle Scholar
  67. 67.
    Mandava NB, Ito Y (1982) Separation of plant hormones by countercurrent chromatography. J Chromatogr 247:315–325CrossRefGoogle Scholar
  68. 68.
    Mandala SM, Thornton RA, Frommer BR, Curotto JE, Rozdilsky W, Kurtz MB et al (1995) The discovery of australifungin, a novel inhibitor of sphinganine N-acyltransferase from Sporormiella australis. Producing organism, fermentation, isolation, and biological activity. J Antibiot 48:349–356PubMedGoogle Scholar
  69. 69.
    Stierle DB, Stierle AA, Ganser B (1997) New phomopsolides from a Penicillium sp. J Nat Prod 60:1207–1209PubMedCrossRefGoogle Scholar
  70. 70.
    Jarvis BB, De Silva T, McAlpine JB, Swanson SJ, Whittern DN (1992) New trichoverroids from Myrothecium verrucaria isolated by high speed countercurrent chromatography. J Nat Prod 55:1441–1446PubMedCrossRefGoogle Scholar
  71. 71.
    Hochlowski JE, Mullally MM, Spanton SG, Whittern DN, Hill P, McAlpine JB (1993) 5-N-acetylardeemin, a novel heterocyclic compound which reverses multiple drug resistance in tumor cells. II. Isolation and elucidation of the structure of 5-N-acetylardeemin and two congeners. J Antibiot 46:380–386PubMedGoogle Scholar
  72. 72.
    Williams RG (1985) Analytical chemistry – applied spectroscopy section, abstract 300. In: Pittsburgh Conference and Exposition, New Orleans, LAGoogle Scholar
  73. 73.
    Breinholt J, Ludvigsen S, Rassing BR, Rosendahl CN, Nielsen SE, Olsen CE (1997) Oxysporidinone: a novel antifungal, N-methyl-4-hydroxy-2-pyridinone from Fusarium oxysporum. J Nat Prod 60:33–35CrossRefGoogle Scholar
  74. 74.
    Rasmussen RR, Scherr MH, Whittern DN, Buko AM, McAlpine JB (1987) Coloradocin, an antibiotic from a new Actinoplanes. II. Identity with luminamicin and elucidation of structure. J Antibiot 40:1383–1393PubMedGoogle Scholar
  75. 75.
    Omura S, Iwata R, Iwai Y, Taga S, Tanaka Y, Tomoda H (1985) Luminamicin, a new antibiotic. Production, isolation and physico-chemical and biological properties. J Antibiot 38:1322–1326PubMedGoogle Scholar
  76. 76.
    Gouda H, Sunazuka T, Ui H, Handa M, Sakoh Y, Iwai Y et al (2005) Stereostructure of luminamicin, an anaerobic antibiotic, via molecular dynamics, NMR spectroscopy, and the modified Mosher method. Proc Natl Acad Sci USA 102:18286–18291PubMedCrossRefGoogle Scholar
  77. 77.
    McAlpine JB, Tuan JS, Brown DP, Grebner KD, Whittern DN, Buko A et al (1987) New antibiotics from genetically engineered actinomycetes. I. 2-Norerythromycins, isolation and structural determinations. J Antibiot 40:1115–1122PubMedGoogle Scholar
  78. 78.
    Hochlowski JE, Hill P, Whittern DN, Scherr MH, Rasmussen RR, Dorwin SA et al (1994) Aselacins, novel compounds that inhibit binding of endothelin to its receptor. II. Isolation and elucidation of structures. J Antibiot 47:528–535PubMedGoogle Scholar
  79. 79.
    Mitchell SS, Whitehall AB, Trapido-Rosenthal HG, Ireland CM (1997) Isolation and characterization of 1,3-dimethylisoguanine from the Bermudian sponge Amphimedon viridis. J Nat Prod 60:727–728PubMedCrossRefGoogle Scholar
  80. 80.
    Hallock YF, Manfredi KP, Dai JR, Cardellina JH 2nd, Gulakowski RJ, McMahon JB et al (1997) Michellamines D-F, new HIV-inhibitory dimeric naphthylisoquinoline alkaloids, and korupensamine E, a new antimalarial monomer, from Ancistrocladus korupensis. J Nat Prod 60:677–683PubMedCrossRefGoogle Scholar
  81. 81.
    Abbott T, Peterson R, McAlpine J, Tjarks L, Bagby M (1989) Comparing centrifugal countercurrent chromatography, nonaqueous reversed phase HPLC and Ag+ ion exchange HPLC for the separation and characterization of triterpene acetates. J Liq Chromatogr 12:2281–2301Google Scholar
  82. 82.
    McAlpine JB, Theriault RJ, Grebner KD, Hardy DJ, Fernandes PB (1987) Minor products from the microbial transformation of 6-O-methylerythromicin A by Mucor circinelloides. In: 27th Interscience conference on antimicrobial agents and chemotherapy. New York, Abstract 222Google Scholar
  83. 83.
    Hochlowski JE, Whittern DN, Hill P, McAlpine JB (1994) Dorrigocins: novel antifungal antibiotics that change the morphology of ras-transformed NIH/3T3 cells to that of normal cells. II. Isolation and elucidation of structures. J Antibiot 47:870–874PubMedGoogle Scholar
  84. 84.
    Kobayashi J, Tsuda M, Fuse H, Sasaki T, Mikami Y (1997) Halishigamides A-D, new cytotoxic oxazole-containing metabolites from Okinawan sponge Halichondria sp. J Nat Prod 60:150–154CrossRefGoogle Scholar
  85. 85.
    Bruening RC, Oltz EM, Furukawa J, Nakanishi K, Kustin K (1986) Isolation of tunichrome B-1, a reducing blood pigment of the sea squirt, Ascidia nigra. J Nat Prod 49:193–204PubMedCrossRefGoogle Scholar
  86. 86.
    Zhang T (1984) Horizontal flow-through coil planet centrifuge: some practical applications of countercurrent chromatography. J Chromatogr 315:287–297CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • James B. McAlpine
    • 1
  • J. Brent Friesen
    • 1
  • Guido F. Pauli
    • 1
  1. 1.Department of Medicinal Chemistry and Pharmacognosy, School of PharmacyUniversity of Illinois at ChicagoChicagoUSA

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