strategies of solvent system selection for the isolation of flavonoids by countercurrent...
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Strategies of solvent system selection forthe isolation of flavonoids by countercurrentchromatography
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
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Introduction
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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
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Introduction Questions…
Is there anySystematic method development for CCC ?
Faster Effective logical
HPLC
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Strategies
1. Choose solvent family
2. Change the ratio & add some modifier
3. Operation ( In Consideration of elution mode )
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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
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1. Choose solvent family
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Strategies of solvent system selection
Solvent families
HEMWAT
ChMWAT
EBuWAT
terAcWat
HterAcWat
Ito et al.
Oka et al.
Margraff et al.
ARIZONA(HpEMWAT)
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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
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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
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Strategies of solvent system selection
HydrophilicLipophilic
HEMWAT
ChMWAT
EBuWAT
terAcWat
HterAcWat
ARIZONA(HpEMWAT)
Based on GUESS mixture polarity
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Strategies of solvent system selection
1. Solubility1. 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)
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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 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…
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2. Change the ratio or add some modifier[ estimate K value ]
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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
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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
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EECCC can help you to survey wide-range of polarity.
Strategies of solvent system selection
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Strategies of solvent system selection
K value estimation
Shake-flask
TLC
Analytical CS
HPLC GC UV
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parallel 2VC EECCC experiments Anal. Chem. 2009, 81, 4048–4059
VR = VM + KDVS
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3. Operation ( In Consideration of elution mode )
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Practical differences are exist
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Strategies of solvent system selection
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CCC for FlavonoidsFocused on solvent systems
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Class of flavonoid
Flavonoid
Free flavonoid
Glycosylated flavonoid
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CCC equipments used for the sepa-ration of flavonoids HSCCC
CPCDCCCRLCC
Literature study
Free formGlycosylated form
89%
54.5%
35.5%
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Isolation of Free flavonoid
Hexane EA MeOH Water
More than 60% used HEMWAT system
XlogP approx 3.0
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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
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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
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EA BuOH Water
Isolation of Glycosylated flavonoid
Link !
Alcohol
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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
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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
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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
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Thank you for your attention