Strategies
of
solvent
system
the isolation of flavonoids by
chromatography
selection
for
countercurrent
Journal of separation science
2010, 33, 336–347
2010 8 6 (Fri)
MinSeok Kang
Introduction
Introduction
History of CCC
In the 1940s
In the 1980s
Craig
machine
CPC
In the 1970s
RLCC
DCCC
HSCCC
Introduction
Questions…
Is there any
Systematic method development for CCC ?
Faster
HPLC
Effective
logical
Strategies
1. Choose solvent family
2. Change the ratio & add some modifier
3. Operation ( In Consideration of elution mode )
Strategies of solvent system selection
Settling time – shorter than 30s ( < 60s )
Requirements for
Suitable Solvent systems
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
Ito et al.
ChMWAT
Oka et al.
HEMWAT
Margraff et al.
ARIZONA(HpEMWAT)
terAcWat
EBuWAT
HterAcWat
Strategies of solvent system selection
J. Brent Friesen et al. JOC A 2007 51-59
G.U.E.S.S. mixture combined EECCC was used for understanding various solvent systems.
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
Strategies of solvent system selection
Lipophilic
Hydrophilic
HEMWAT
HterAcWat
EBuWAT
terAcWat
ChMWAT
ARIZONA(HpEMWAT)
Based on GUESS mixture polarity
Strategies of solvent system selection
Unknown or known??
1. Solubility
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
1. Literature study
What you got?
( start with crude sample)
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 ]
Ether
Produce peroxide
AND SO ON…
Many solvent system families are limited
TBME : EA : MeOH : DDW = 5:5:5:5 or more
tertACWAT / increasing A CN ratio
Hex : TBME : A CN : Water = 5:5:5:5 or more
Single phase
Single phase
Three phase …but it could be used for CCC
2. Change the ratio or add some modifier
[ estimate K value ]
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
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
Strategies of solvent system selection
EECCC can help you to survey wide-range of polarity.
Strategies of solvent system selection
K value estimation
Shake-flask
HPLC
GC
Analytical CS
TLC
UV
parallel 2VC EECCC experiments
VR = VM + KDVS
Anal. Chem. 2009, 81, 4048–4059
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
Free flavonoid
Flavonoid
Glycosylated flavonoid
CCC equipments used for the separation of
flavonoids
89%
HSCCC
CPC
DCCC
RLCC
Literature study
35.5%
54.5%
Free form
Glycosylated form
Isolation of Free flavonoid
XlogP approx 3.0
More than 60% used HEMWAT system
Hexane
EA
MeOH
Water
Isolation of Free flavonoid
Modifier
Hexane
EA
MeOH
CHCl3
EtOH
Alter aqueous phase
CH2Cl2
PrOH
Alter both phase
BuOH
Alter organic phase
Polarity wil be lowered
Recommended when sample is more
soluble in chlorinated solvents than
in EA
Water
Isolation of Glycosylated flavonoid
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
Isolation of Glycosylated flavonoid
EA
BuOH
Alcohol
Link !
Water
Isolation of Glycosylated flavonoid
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
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
The addition of BuOH to the system
( As Organic modifier )
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