Future and potential of Countercurrent Chromatography (CCC) from preparative isolation of compounds to the production of Knock-out Extracts. Can CCC become a mainstream technique?

1. The Future of K-Based Chromatography
The 9th International CCC Event in Chicago/USA
Conference: August 1-3, Workshop: July 30-31, 2016
Dominican University, River Forest, IL (U.S.A.)

2. CCS as a “niche” technique
• Preparative isolation of known compounds
HO
HO
C
O
OH
HO
HO
H
C
C
H
C
O
OH
HO
HO
H
C
C
H
C
O
O
OH
OH
HO C
O
OH

3. CCS as a “niche” technique
• Specialized method of performing separations already
done with column chromatography methods.
HO
HO
H
C
C
H
C
O
OH
HO
HO
H
C
C
H
C
O
O
OH
OH
HO C
O
OH

4. Countercurrent Separations
Into the Mainstream
A wok in every kitchen and
http://lh4.googleusercontent.com/public/l33bGiAu5pLcwwuK9O68cynPISlfGvpyWbYWUj-Vrqqt70OIrBK8in8xcMUa0lP40mZij6vDSPa381L7sOO16GqzZbCWcmmKdDu_Mr7FQR9JBT0O_sr0kSiZZEjlq3cQ__Xq9qheN3hSTFbC-wLR0qmzthl4JxQvlb0PX9XUQz7m29bUW1E
a CCS instrument in every laboratory

5. What is necessary to make CCS a
“mainstream” technique?

6. What is necessary to make CCS a
“mainstream” technique?
A. Purify and characterize low concentration metabolites
3. Multiple step isolation schemes that integrate CCS
2. Sequential CCS runs
B. Innovations that push the limits of the technology
C. Emphasize the importance of K
1. Enrichment Strategies
D. Applications of CCS to Discovery

7. http://www.interfaces.com/blog/wp-content/uploads/2013/05/Divergent-Road-with-low-hanging-fruit.jpg
A. Isolation of low concentration compounds will
lead to new compounds

8. new chalcones 4, 11, & 12
previously unreported in hops:
5, 6, 9b, & 13
Estrogens and Congeners from Spent Hops
(Humulus lupulus)
Lucas R. Chadwick, Dejan Nikolic, Joanna E.
Burdette, Cassia R. Overk, Judy L. Bolton, Richard
B. van Breemen, Roland Frohlich, Harry H. S. Fong,
Norman R. Farnsworth, and Guido F. Pauli
J. Nat. Prod. 2004, 67, 2024-2032
http://www.aspca.org/~/media/Files/pet-care/poison-control/plants/large-images/hops-1.ashx
A. Purify and characterize unknown analytes

9. A. Purify and characterize
low concentration analytes
Enrichment and identification of ginkgotoxin
Quantitative (400 MHz) 1H-NMR analysis
(a) the extract of a single seed;
(b) the targeted HSCCC fraction
corresponding to a K value of 1.62
(ChMWat, 10:5:5);
(c) synthetic
Liu, Y.; Chen, S. N.; McAlpine, J. B.; Klein, L. L.; Friesen, J. B.; Lankin, D. C.; Pauli, G. F., Quantification of a Botanical
Negative Marker without an Identical Standard: Ginkgotoxin in Ginkgo biloba. Journal of Natural Products 2014, 77, 611-617
59 µg of ginkgotoxin per seed

10. Alpinia combined fraction in hexane / tert-butylmethylether / methanol / water 5:5:5:5
0 0.25 0.5 0.75 1 1.33 2 4 ¥K'(1)
A
OH
O
Alpinia DCM extract in hexane / ethyl acetate / methanol / water 5:5:5:5
0 0.25 0.5 0.75 1 1.33 2 4 ¥K'(1)
A
0
5
10
15
mg
280nm
230nm
mg
OH
O
OO
HO
OO
O O
OH
O
Orthogonality

11. A. Purify and characterize low concentration metabolites :
2. Sequential CCS runs
CCC Sample Cutting for Isolation of Prenylated Phenolics from Hops
Lucas R. Chadwick, Harry H. S. Fong, Norman R. Farnsworth, and Guido F. Pauli
Journal of Liquid Chromatography & Related Technologiesw, 28: 1959–1969, 2005

12. 3. Multiple step isolation schemes that integrate CCS
Almost a third of the surveyed articles perform a preliminary column
chromatography step prior to the CCS experiment.
column media:
• silica gel
• C-18 functionalized silica gel
• D101
• XAD-7, XAD-2, XAD-4
• Toyopearl TSK HW-50(F)
• AB-8
• Sephadex LH-20
• polyamide
preparative steps:
§ solid-liquid extraction
§ liquid-liquid extraction
§ precipitation
§ flash chromatography
A. Purify and characterize low concentration metabolites :
Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of
Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.

13. What is necessary to make CCS a
“mainstream” technique?
A. Purify and characterize low concentration metabolites
B. Innovations that push the limits of the technology
C. Emphasize the importance of K
1. Off/online Detection: UV, ELSD, MS, & NMR
2. Knockout strategies for biological testing.
D. Applications of CCS to Discovery

14. B.1. GC shows phytochemical complexity
crude
2 major
16 minor
compounds
fraction #3
out of 6
60
compounds
refractionation
of #3
600
compounds

15. Silymarin HSCCC separation, HChMWat/AcOH (1:20:22:12:12)
15
Isosilybins A+B
Silybins A+B
α,β–cis
Sily-compounds
Silydianin
Silychristins
Isosilychristins
Taxifolin
Silydianin-like
compound
Aromadendrin
1
H NMR spectra (600 MHz, DMSO-d6), 3.4–5.5 ppm.
B.1.

16. B.2. Innovations that push the limits of the technology
http://heartcurrents.files.wordpress.com/2010/03/wp_heart_fresh-cranberry-juice-12614054901.jpg http://www.chemistry-blog.com/wp-content/uploads/2008/10/column.jpg
active
fractions
Diaion HP-20
active fractions
- benzoic acid
An experimental implementation of chemical subtraction
Journal of Pharmaceutical and Biomedical Analysis 46 (2008) 692–698
Shao-Nong Chen, Allison Turner, Birgit U. Jaki, Dejan Nikolic, Richard B. van Breemena, J. Brent Friesen,
Guido F. Pauli

17. Total Extract (TE)
K-Targeted
Metabolites (Ts)
DESIGNER Extract
(Ts-DE)
Difference ExtractTotal Extract K-Targeted
Metabolites
Enriched
Depleted
Extract
Extract
Ramos Alvarenga, R. F.; Friesen, J. B.; Nikolic, D.; Simmler, C.; Napolitano, J. G.; vanBreemen, R.; Lankin, D. C.;
McAlpine, J. B.; Pauli, G. F.; Chen, S. N., K-Targeted Metabolomic Analysis Extends Chemical Subtraction to DESIGNER
Extracts: Selective Depletion of Extracts of Hops (Humulus lupulus). Journal of Natural Products 2014, 77, 2595-2604.

18. Total Extract HEMWat 0
IX
IX-PDE
HterAcWat +3
IX
IX-PDE
IX-DE
Total Extract HEMWat 0
8-PN
6-PN
XH
HEMWat -3
8-PN/6-PN
8-PN/6-PN-PDE
8-PN/6-PN-DE8-PN/6-PN-PDE
Total Extract HEMWat 0
XH
XH-PDE
HEMWat -3
XH
XH-PDE
IX-DE
IX HterAcWat +3Total Extract HEMWat 0
8-PN
6-PN
XH
HEMWat -3
8-PN/6-PN
MultiT-PDE
XH
MultiT-DE
MultiT-PDE
MultiT-PDE
IX
Knockout
Extraction
Schemes
Ramos Alvarenga, R. F.; Friesen,
J. B.; Nikolic, D.; Simmler, C.;
Napolitano, J. G.; vanBreemen,
R.; Lankin, D. C.; McAlpine, J.
B.; Pauli, G. F.; Chen, S. N., K-
Targeted Metabolomic Analysis
Extends Chemical Subtraction to
DESIGNER Extracts: Selective
Depletion of Extracts of Hops
(Humulus lupulus). Journal of
Natural Products 2014, 77,
2595-2604.
B.2. Innovations that push the limits of the technology

19. What is necessary to make CCS a
“mainstream” technique?
A. Purify and characterize low concentration metabolites
B. Innovations that push the limits of the technology
C. Emphasize the importance of K
D. Applications of CCS to Discovery
1. Reciprocal Symmetry Plots
2. Monitoring Eluent Phase Composition

20. 2 3
20
69
184
247
99
26
16
0
50
100
150
200
250
300 K value distribution
#ofcompounds
1 1
11
19
3
1
0
5
10
15
20
# compounds
K value
K values of compounds for articles
isolating only one compoundC. Emphasize the
importance of K
Friesen, J. B.; McAlpine, J. B.; Chen, S.-N.; Pauli, G. F., Countercurrent Separation of
Natural Products: An Update. Journal of Natural Products 2015, 78, 1765-1796.

21. Model Compounds:
HO
H
H H
H
O
O
OH
OH
O
O O
HO
H H
H
CH3
OH
OH O
OHO
OH
N
O
OH
O
OH
O
HO
O
O
OH
O OHO
O
H
HO
H
HO
H
H
OHH
O
OH
OH
O
OH
O
O
OH OH
OH
O
O
OH
OH
HO
O
O
H
HO
H
HO
H
H
OHH
O
OH
OH
N
N
N
N
O
O
N
H
O
OH
NH2
N
N
OH
S
O
O
O
SO
O
O
S
O
O
O
3Na
N
H
N
O
OH
H
H
O
O
O
O
O
O
O
O
OH
OH
OH
OOH
HO
The GUESSmix
Friesen J.B, Pauli G.F. Journal of Liquid
Chromatography and Related Technologies, 28:
2777-2806, 2005
b
O
Q
r
R
U
F
Y
C
I
E
MZ
V
G
T X
H
D
N
A

22. C. Emphasize the
importance of K
Pauli, G.; Pro, S.; Friesen, J. B.
Countercurrent Separation of
Natural Products. J. Nat. Prod.
2008, 71, 1489-1508
Using the
GUESSmix for
method
development

23. C. Emphasize the importance of K
Using GUESSmix to explore solvent system families.
Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007)
Symmetry
Midline
0
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ¥K
A
M
Q
V
U
F
N
Z E

24. C.1. K-based chromatograms
• The “sweet spot” present in CCS separations that is related to
the distribution constant (K = Cstat/Cmob) of a certain compound in a
particular solvent system.
• The key to CCS separations is putting the target compound(s) in
the sweet spot where optimal separation can take place.
GUESSmix in HEMWat 4646
0
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 34 5.33 8 16
¥K
A
280nm
230nm
Reciprocal Symmetry Plots

25. The GUESSmix in HEMWat 4646
0
0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425mL
A
280nm
230nm
Reciprocal Symmetry (ReS and ReSS) Plots
have 0 £ K £ ¥ on the x axis.
Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007)
M
Q
V
U
F
N
Z E
Symmetry
Midline
0
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ¥K
A
M
Q
V
U
F
N
Z E

26. HEMWat +5 3737
-0.01
0 0.5 1 1.5 2 2.67 4 8 ¥K'(2)
A
280nm
230nm
HEMWat +3 4646
-0.01
0 0.5 1 1.5 2 2.67 4 8K'(2)
A
280nm
230nm
HEMWat +4 3746
-0.01
0 0.5 1 1.5 2 2.67 4 8K'(2)
A
280nm
230nm
HEMWat Systems I
M
M
M
Q
Q
Q
Z
Z
Z
U
U
U
F
F
F
¥
¥
C.1. ReS and ReSS plots
Friesen, J. B.; Pauli, G. F., Journal of Agricultural and Food Chemistry 2008, 56, 19-28

27. HEMWat 0 5555
-0.01
0 0.5 1 1.5 2 2.67 4 8K'(2)
A
280nm
230nm
HEMWat +2 3755
-0.01
0 0.5 1 1.5 2 2.67 4 8 ¥K'(2)
A
280nm
230nm
HEMWat +1 4655
-0.01
0 0.5 1 1.5 2 2.67 4 8K'(2)
A
280nm
230nm
HEMWat Systems II
M
Z
F
F
F
U
U
U
M
M
Q
Q
Q
Z
Z
¥
¥
C.1. ReS and ReSS plots
Friesen, J. B.; Pauli, G. F., Journal of Agricultural and Food Chemistry 2008, 56, 19-28

28. C.1. ReS and ReSS plots
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ∞
KD
A
280nm
230nm
I II III
r
C
F
U
V
M
Q
N
Z
E
O
b
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ∞
KD
A
I II III
MSrun time = 7.2 hours
run time = 6.3 hours
Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007)

29. 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ∞
KD
A
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.29 2.67 3.2 4 5.33 8 16 ∞
KD
A
I II III
I II III
run time = 4.8 hours
run time = 5.8 hours
C.1. ReS and ReSS plots
Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007)

30. C. Emphasize
the importance of K
CCC Sample Cutting for Isolation of Prenylated Phenolics from Hops
Lucas R. Chadwick, Harry H. S. Fong, Norman R. Farnsworth, and Guido F. Pauli
Journal of Liquid Chromatography & Related Technologiesw, 28: 1959–1969, 2005

31. C.2. Monitoring Eluent Phase Composition
0
0.5
1
0
50
100
25 50 75 100 125
Sf value
K value
minutes
pma value
K value
Sf value
0
0.2
0.4
0.6
0.8
1
299,500
300,500
301,500
302,500
303,500
0 20 40 60 80 100 120
PMA
values
time in min
PMA
UV
Sf
K/10
Pauli, G. F.; Pro, S. M.; Chadwick, L. R.; Burdick, T.; Pro, L.; Friedl, W.; Novak, N.; Maltby, J.; Qiu, F.; Friesen, J. B., Real-Time Volumetric
Phase Monitoring: Advancing Chemical Analysis by Countercurrent Separation. Analytical Chemistry 2015, 87, 7418-7425.
ChMWat
LP mobile
EECCC
Automated Separation of Green Tea Catechins .
terAcWat 5:5:10 solvent system with lower phase mobile.0.5
mL/mn flow rate, 2,000 rpm, 0.65 Sf , 20 oC.

32. D. Applications of CCS to discovery
1. Isolation of target compounds
2. bioassy guided fractionation
3. metabolomic studies
Find a single fish in the sea
Identify compounds with certain biological activity
Identify as many compounds as possible from a single source

33. D. Applications of CCS to discovery
2. bioassay guided fractionation
0 0.5 1 1.5 2 2.5 3 3.5 4 4.57 5.33 6.4 8 10.67 16 32 ¥
Oplopanax crude extract in hexane / DCM / methanol / water 7:3:7:3
hexane / ethyl acetate 8:2 hexane / ethyl acetate 7:3
(1) neroplomacrol (novel)
(2) neroplofurol (novel)
(3) oplopandiol
(4) falcarindiol
(5) sesamin
Sesquiterpenes from Oplopanax horridus
Taichi Inui,Yuehong Wang, Dejan Nikolic, David C. Smith, Scott G.
Franzblau, and Guido F. Pauli
J. Nat. Prod. 2010, 73, 563–567
http://www.wnps.org/plants/oplopanax_horridus.html

34. Advanced applications of
counter-current chromatography
in the isolation
of anti-tuberculosis constituents
from Dracaena angustifolia
Ryan J. Case, Yuehong Wang, Scott G.
Franzblau, D. Doel Soejarto,
Lohi Matainaho, Pius Piskaut, Guido F. Pauli
Journal of Chromatography A,
1151 (2007) 169–174
D. Applications of CCS to discovery
2. bioassay guided fractionation
http://210.240.49.194/~wonder/plant/plant91/plant91-11/plt91-11-3-7.htm

35. Biochromatogram
Alpinia DCM extract in HEMWat 5/5/5/5
0
30 40 50 60 70 80 90 100 110 120Tube
A
0
20
40
60
80
100
280nm
230nm
% inhibition at 64 ug/ml
%
Alpinia DCM extract in HEMWat 5/5/5/5
0
30 40 50 60 70 80 90 100 110 120Tube
A
0
5
10
15
mg
280nm
230nm
mg
MIC
1.8-0.9
307.5
O
O
O
O
ACA

36. redistribution
of anti-TB
activity
Alpinia DCM extract in HEMWat 5/5/5/5
0
40 42 44 46 48 50 52 54Tube
A 0
20
40
60
80
100
280nm
230nm
% inhibition at 64 ug/ml
%
Alpinia Fraction 3, H/tBME/M/Wat 5:5:5:5
0
15 25 35 45 55 65 75 85 95 105 115tube
A
0
20
40
60
80
100
%
230nm
280nm
% inhibition

37. D.2. Bioassay-Guided Fractionation
Fig. 5. Purification of the subfractions 24.4 and 24.5 using analytical HSCCC.
Instrument: analytical HSCCC TBE-20; coil column volume: 16 ml; flow rate:
0.3 ml/min; sample loaded: 18.12mg in 200l diphase solvent; diphase solvent
system:HEMW (5.25:5:5.25:5); stationary phase: upper phase; mobile phase:
lower phase.
Talanta. 2010 Sep 15;82(4):1521-7. doi: 10.1016/j.talanta.2010.07.036. Quick identification of
apoptosis inducer from Isodon eriocalyx by a drug discovery platform composed of analytical
high-speed counter-current chromatography and the fluorescence-based caspase-3 biosensor
detection. Han QB, Yu T, Lai F, Zhou Y, Feng C, Wang WN, Fu XH, Lau CB, Luo KQ, Xu HX,
Sun HD, Fung KP, Leung PC.

38. Fig. 3. Effects of VOAS fractions from solvent system 8 (heptane–ethyl acetate–methanol–water
at a volume ratio of 27:23:27:23%) on HUVEC viability. HUVECs were treated with (A) VOAS
fraction groups (three consecutive fractions combined into one group) at 1:100 dilution and (B)
individual fractions at 1:100 dilution from active fraction groups. MTS assay was performed after
48 h of incubation. Data are expressed as % of medium control and are mean ± SD. *Significant
inhibitory effects compared to vehicle control (Veh, DMSO 1%) (P < 0.05, unpaired Student’s t-
test). VOAS at 10, 33 and 100 g/mL was used as an inhibitory control. The results demonstrated
that fraction groups III–VI and fractions 7–15 exhibited significant anti-endothelial properties.
J Chromatogr A. 2012 May 4;1236:132-138. doi: 10.1016/j.chroma.2012.03.013. Bioactivity-
guided fractionation of the volatile oil of Angelica sinensis radix designed to preserve the
synergistic effects of the mixture followed by identification of the active principles.
Yeh JC, Garrard IJ, Cho CW, Annie Bligh SW, Lu GH, Fan TP, Fisher D.
D.2. BAGF

39. D. Applications of CCS to discovery
Phytoconstituents from Vitex agnus-castus Fruits
Chen SN, Friesen JB, Webster D, Nikolic D, van Breemen RB, Wang
ZJ, Fong HH, Farnsworth NR, Pauli GF.
Fitotherapia, in press 2011,
70 kg Fruit
defat with petroleum ether and extract with methanol (879 g)
Diaion HP-20 flash chromatography (5 fractions)
Silica Gel Vacuum-Liquid Chromatography (13 fractions)
Sephadex LH-20 CC (4 fractions)
HSCCC (11 fractions)
HPLC YMC ODS-AQ HPLC YMC C-18
(21) p-coumaric acid
(22) 4-hydroxybenzoic acid
(23) ficusal
(24) vladirol
(25) balanophonin
24 compounds total
1 new compound
3. metabolomic studies

40. Fig. 2. Deconvolved biochromatogram of O. horridus crude extract. The x-axis represents the K
values of the CCC fractions, and the y-axis indicates the anti-TB activities of the fractions. The
bioactivities for all fractions observed at 50 μg/ml (black line) were deconvolved into Gaussian
peaks to produce 19 biopeaks (colored), which are representative of the individual active
principles and thus were used for Pearson's correlation. Each of the 19 biopeaks contains
multiple compounds which were further analyzed by GC–MS.
Fig. 6. Distribution of the 24 anti-TB active metabolites in the 3D CCC–GC–
Pearson matrix. The assigned peaks are color coded in accord to the structural
classes: pink for polyketides, green for cadinol type sesquiterpenes, blue for
ledol type sesquiterpenes, and brown for others.
Fitoterapia. 2012 Oct;83(7):1218-25. doi: 10.1016/j.fitote.2012.06.012.
Unbiased evaluation of bioactive secondary metabolites in complex matrices.
Inui T1, Wang Y, Pro SM, Franzblau SG, Pauli GF.
D.3. metabolomic studies