Strategies of solvent system selection for the isolation of flavonoids by countercurrent chromatography (Fri) MinSeok Kang Journal of separation science 2010, 33, 336–347

Introduction

In the 1940s In the 1970s History of CCC Craig machine RLCCDCCC In the 1980s CPC HSCCC

IntroductionQuestions… Is there any Systematic method development for CCC ? Faster Effective logical HPLC

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

Strategies of solvent system selection Requirements for Suitable Solvent systems Settling time – shorter than 30s ( < 60s ) Partition coefficient – 0.5 < K < 2.0 Separation factor – greater than 1.5 Solvent system should produce similar volumes for each phase

1. Choose solvent family

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

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

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

Strategies of solvent system selection HydrophilicLipophilic HEMWAT ChMWAT EBuWAT terAcWat HterAcWat ARIZONA(HpEMWAT) Based on GUESS mixture polarity

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

Strategies of solvent system selection CCC user should notice solvent properties. 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 Hex : TBME : A CN : Water = 5:5:5:5 or more Three phase … but it could be used for CCC EA can be… Hydrolysis by water Produce EtOH and acetic acid CHCL3 can be…Acidified ( turn into HCl ] EtherProduce peroxide AND SO ON…

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

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

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

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

K value estimation Shake-flask TLC Analytical CS HPLC GC UV

parallel 2VC EECCC experiments Anal. Chem. 2009, 81, 4048–4059 V R = V M + K D V S

3. Operation ( In Consideration of elution mode )

Practical differences are exist

Strategies of solvent system selection

CCC for Flavonoids Focused on solvent systems

Class of flavonoid Flavonoid Free flavonoid Glycosylated flavonoid

89% 54.5% 35.5%

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

Modifier Hexane EA MeOH Water Isolation of Free flavonoid EtOH PrOH BuOH CHCl 3 CH 2 Cl 2 Alter aqueous phase Alter both phase Alter organic phase Polarity wil be lowered Recommended when sample is more soluble in chlorinated solvents than in EA

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

EA BuOH Water Isolation of Glycosylated flavonoid Link ! Alcohol

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

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

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

Thank you for your attention