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2010 8 6 (Fri) MinSeok Kang

Strategies of solvent system selection for the isolation of flavonoids by countercurrent chromatography. Journal of separation science 2010, 33, 336–347. 2010 8 6 (Fri) MinSeok Kang . Introduction. Introduction. History of CCC. In the 1940s . Craig machine. In the 1980s .

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2010 8 6 (Fri) MinSeok Kang

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  1. Strategies of solvent system selection forthe isolation of flavonoids by countercurrentchromatography Journal of separation science 2010, 33, 336–347 2010 8 6 (Fri) MinSeok Kang

  2. Introduction

  3. Introduction History of CCC In the 1940s Craig machine In the 1980s In the 1970s HSCCC CPC RLCC DCCC

  4. Introduction Questions… Is there any Systematic method development for CCC ? Faster Effective logical HPLC

  5. Strategies 1. Choose solvent family 2. Change the ratio & add some modifier 3. Operation ( In Consideration of elution mode )

  6. Strategies of solvent system selection

  7. 1. Choose solvent family

  8. Strategies of solvent system selection Solvent families Ito et al. ChMWAT Oka et al. HEMWAT Margraff et al. ARIZONA(HpEMWAT) terAcWat EBuWAT HterAcWat

  9. J. Brent Friesen et al. JOC A 2007 51-59 Strategies of solvent system selection G.U.E.S.S. mixture combined EECCC was used for understanding various solvent systems.

  10. Strategies of solvent system selection EBuWAT terAcWat terAcWat 6:4:10 HterAcWat 4:6:4:6 Each solvent system has somewhat restriction. J.B. Friesen / J.Chromatogr. A 1151(2007) 51-69

  11. Strategies of solvent system selection Lipophilic Hydrophilic HEMWAT EBuWAT HterAcWat terAcWat ChMWAT ARIZONA(HpEMWAT) Based on GUESS mixture polarity

  12. Strategies of solvent system selection Unknown or known?? 1. Solubility 1. Literature study What you got? ( start with crude sample) What solvent can your target dissolve? Where is your target come from? ( Liq-Liq partition ) 2 Co-TLC with GUESS Assuming polarity of your target 3. Literature study

  13. Strategies of solvent system selection CCC user should notice solvent properties. EA can be… Hydrolysis by water Produce EtOH and acetic acid CHCL3 can be… Acidified ( turn into HCl ] AND SO ON… Ether Produce peroxide Many solvent system families are limited TBME : EA : MeOH : DDW = 5:5:5:5 or more Single phase tertACWAT / increasing A CN ratio Single phase Three phase …but it could be used for CCC Hex : TBME : A CN : Water = 5:5:5:5 or more

  14. 2. Change the ratio or add some modifier [ estimate K value ]

  15. Strategies of solvent system selection It has a dog’s chance Non-polar direction User need to get started with 5:5:5:5 Polar direction

  16. Strategies of solvent system selection 10:17:3 10:17:8 10:17:10 Petroleum ether (60–90C):ethanol:water in volume ratios F. Yang et al. / J. Chromatogr. A 829 (1998) 137 –141

  17. Strategies of solvent system selection EECCC can help you to survey wide-range of polarity.

  18. Strategies of solvent system selection K value estimation Shake-flask Analytical CS HPLC GC TLC UV

  19. parallel 2VC EECCC experiments Anal. Chem. 2009, 81, 4048–4059 VR = VM + KDVS

  20. 3. Operation ( In Consideration of elution mode )

  21. Practical differences are exist

  22. Strategies of solvent system selection

  23. CCC for Flavonoids Focused on solvent systems

  24. Class of flavonoid Free flavonoid Flavonoid Glycosylatedflavonoid

  25. 89% 35.5% 54.5%

  26. Isolation of Free flavonoid XlogP approx 3.0 More than 60% used HEMWAT system Hexane EA MeOH Water

  27. Isolation of Free flavonoid Modifier Hexane EA MeOH Water CHCl3 EtOH Alter aqueous phase CH2Cl2 PrOH Alter both phase Polarity wil be lowered Recommended when sample is more soluble in chlorinated solvents than in EA BuOH Alter organic phase

  28. Isolation of Glycosylatedflavonoid • XlogP approx more than 4.0 More than 60% used EbuWAT & Chemwat system The exist of sugar moiety enhances its polarity. More polar solvent system needed. EA BuOH Water

  29. Isolation of Glycosylatedflavonoid EA BuOH Water Alcohol Link !

  30. Isolation of Glycosylatedflavonoid Key factor “ BEST solvent “ CHCl3 MeOH Water Alcohol But In case of PrOH, some limitations Settling time is higher than 30s and emulsion occured MtBE BuOH ACN Water(TFA) Especially , used in 70% of the case where anthocyanin pigments Organic modifier Aqueous modifier

  31. Isolation of Mixtures 1. Isocratic mode EtOAc-MeOH-H2O EtOAc-BuOH-H2O CHCl3-MeOH-H2O HEMWAT 2. Gradient mode CHCl3-MeOH-H2O 4:3:2 was used for the isolation of the less polar isoflavones. Hex-EtOAc-MeOH-H2O 0.6:4:0.05:1 Hex-EtOAc-MeOH-H2O 0.6:4:0.7:1 The addition of BuOH to the system ( As Organic modifier )

  32. Conclusion 1. CCC-chromatographer should consider suitable solvent systems before start to separate natural compound 2. Some solvent systems have been introduced to separate specific compound 3. As you can add some modifier or change system mode , you can give some variation protocol. 4. Separation for flavonoid using CCC could be archived by HEMWAT & CHeMWAT system

  33. Thank you for your attention

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