Strategies of solvent system selection forthe isolation of flavonoids by countercurrentchromatography

2010 8 6 (Fri) MinSeok Kang

Journal of separation science 2010, 33, 336–347

Introduction

Mix-ing

Sepa-rating

Set-tling

Introduction

In the 1940s

In the 1970s

History of CCC

Craig ma-chine

RLCC DCCC

In the 1980s

CPC HSCCC

Introduction Questions…

Is there anySystematic 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 Suit-able Solvent systems

Settling time – shorter than 30s ( < 60s )

Partition coefficient – 0.5 < K < 2.0Separation 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 sys-tems.

J. Brent Friesen et al. JOC A 2007 51-59

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

HydrophilicLipophilicHEMWAT

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 phasetertACWAT / increasing A CN ra-tio

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

acidCHCL3 can be…

Acidified ( turn into HCl ]

Ether Produce 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 ra-tios

EECCC can help you to survey wide-range of polarity.

Strategies of solvent system selection

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

VR = VM + KDVS

3. Operation ( In Consideration of elution mode )

Practical differences are exist

Strategies of solvent system selection

CCC for FlavonoidsFocused on solvent systems

Class of flavonoid

Flavonoid

Free flavonoid

Glycosylated flavonoid

CCC equipments used for the sepa-ration of flavonoids HSCCC

CPCDCCCRLCC

Literature study

Free formGlycosylated form

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

CHCl3

CH2Cl2

Alter aqueous phase

Alter both phase

Alter organic phasePolarity 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 modifier Aqueous modifier

Isolation of Mixtures

1. Isocratic mode

2. Gradient mode

EtOAc-MeOH-H2O EtOAc-BuOH-H2O CHCl3-MeOH-H2O

HEMWAT

CHCl3-MeOH-H2O 4:3:2was 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 sys-tems 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