The workshop presented by Guy H. Harris focuses on integrating countercurrent chromatography (CCC) into natural product purification workflows, emphasizing systematic solvent system selection and the importance of distribution ratios. It discusses the benefits and considerations of various CCC instrumentation types and methodologies used in bioassay-guided natural product isolation. Key processes include resolution, purification, and polishing steps tailored for isolating biologically active compounds from complex mixtures.

1. Integrating Countercurrent
Separations into Natural Product
Purification Workflows
Countercurrent Chromatography Workshop
Amercian Society of Pharmacognosy Annual Meeting
Lexington, Kentucky
July 21, 2018
Guy H. Harris, PhD
Independent Consultant
Asbury, New Jersey
1

2. Integrating Countercurrent
Separations into Natural Product
Purification Workflows
or… “How and Why to use CS when
you don’t have too!”
Countercurrent Chromatography Workshop
Amercian Society of Pharmacognosy Annual Meeting
Lexington, Kentucky
July 21, 2018
Guy H. Harris, PhD
Independent Consultant
Asbury, New Jersey 2

3. Systematic Solvent System
Selection
Process Incorporation
“Almost is good enough”
G,Harris ASP CS Workshop, July 21, 2018
3

4. Getting Started Quickly = Solvent Systems - Kd - Sf
1) The most critical parameters are distribution ratio, Kd and stationary phase
retention (stationary phase fraction), Sf
2) Chemistry not instrumentation!
3) Liquid-liquid distribution!
General rules to get started quickly:
1) Almost all literature natural product CS have been accomplished with HEMWat or
CMWat SS series => Great starting (and likely ending) point
o Don’t get too creative to start => remove yourself as a variable J
2) Be systematic in search for SS => HEMWat and CMWat series are predictable
o pH control
3) Minimize SS development by exploiting elution-extrusion (EECCC) using “almost is
good enough” systems developed for particular lab process problems
4) Query scientific literature** for target or related compounds
** The CS scientific literature describing natural products separations is extremely
complicated and inconsistent to sort through (acronyms, terminology, reliability,…)
BUT - the principle behind all is simple => liquid-liquid distribution => the take home from
any paper is the SS
G,Harris ASP CS Workshop, July 21, 2018
4

5. CS Chromatogram in Terms of Kd
0
0.5
1
1.5
2
2.5
0 10 20 30 40 50 60 70 80
Vr (mL)
Column Volume: 17.89mL
Stationary Phase Retention: 70%
Theoretical Plates, N: 750
Vo
Kd=1
Kd=2
Kd=4
Vm
Vc
Calculated for:
Kd = [stationary phase] / [mobile phase]
where Kd = distribution ratio
Vr = Vm + KdVs
Kd = (Vr – Vm)/Vs
where: Vr = retention volume
Vm = mobile phase volume
Vs = stationary phase volume
Sf = Vs / Vc
where Sf = stationary phase fraction
G,Harris ASP CS Workshop, July 21, 2018
5

6. Two Fundamental Types of Modern CCS Instrumentation
Distinguished by the mechanism of phase retention and mixing
Hydrodynamic
“CCC”
Hydrostatic
“CPC”
Ito coil, MLCPC, HPCCC,… “Sanki”, FCPC,…
G,Harris ASP CS Workshop, July 21, 2018 6

7. CCS Instrumentation Considerations
• Huge overlap in applicability of CCC and CPC instrumentation to
most natural product problems
• “Black box” whose purpose is to retain a liquid stationary phase
(= THE COLUMN)
– Purpose of hardware, pumps and CS instrument, is simply to put
the correct liquid in the correct place at the correct time!
– Stationary phase retention
• Primary factors to consider:
– Stationary phase retention, stationary phase retention, stationary
phase retention…..
• Dependent on sample, solvent system, flow rate
– Reliability
G,Harris ASP CS Workshop, July 21, 2018
7

8. HEMWat SS Series
Letter Number Heptane
Ethyl
Acetate Methanol Water
%Water
LP
A 6 0 1 0 1
B 7 1 19 1 19
C 8 1 9 1 9
D 9 1 6 1 6
F 10 1 5 1 5
G 11 1 4 1 4
H 12 1 3 1 3
J 13 2 5 2 5 40
K 14 1 2 1 2
L 15 2 3 2 3 51
M 16 5 6 5 6
N 17 1 1 1 1 64
P 18 6 5 6 5
Q 19 3 2 3 2
R 20 2 1 2 1
S 21 5 2 5 2
T 22 3 1 3 1
U 23 4 1 4 1
V 24 5 1 5 1
W 25 6 1 6 1
X 26 9 1 9 1
Y 27 19 1 19 1
Z 28 1 0 1 0
Composition of phases by numbered system: I.J.Garrard. L.Janaway, D.Fisher, J. Liq. Chrom. Rel. Tech., 30 (2007), 151-163
Solvent systems by letter: ARIZONA
G,Harris ASP CS Workshop, July 21, 2018 8

9. Correlation of Kd and CCS Chromatogram – GUESS Mix
Standards
Relative Compound Polarity
Polar Nonpolar
RelativeSSPolarity
Polar
Non-
polar
C
C
C
A
A
A
F
F
F
N
N
N
40%
H2O
Kd=1
logKd=0
-0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
30 40 50 60 70
logKd
% Water
O
OH
OOH
OH
O
O
OH
O
OH
O
OH
O
N
N
N
N
H
O
O
Caffeine (C) Ferulic acid (F) Aspirin (A) Naringenin (N)
“test tube” Kd
CS Chromatogram
51%
H2O
64%
H2O
G,Harris ASP CS Workshop, July 21, 2018
9

10. Bioassay-Guided Natural Product Isolation as a 3-Step Process
Step Purpose Common Methodology
1 Resolution &
Recovery
• Resolution of multiple
activities
• Recovery from biomatrix
• Localize assay
interferences
• Macroporous resins
• Liquid-liquid extraction
(LLE)
• CS
2 Purification • Separation of active
from inactive
components
• Orthogonal separation
mechanism to steps 1
and 3
• Association of activity
with a specific
component(s)
• Silica gel / diol
• IEX
• Low pressure RP
• CS
3 Polishing • Final purification
• Highest resolution
• RP/NP HPLC
• Crystallization
• CS
Harris, G.H., in The Handbook of Industrial Mycology, ed. Z. An, Marcel Dekker, New York, 2004
G,Harris ASP CS Workshop, July 21, 2018
10

11. P-504062-001X
0
50
100
%MobilePhase
0
500
mVolts
0 10 20 30
Minutes
Zero96_B:196_B:296_B:396_B:496_B:596_B:696_B:796_B:896_B:996_B:1096_B:1196_B:1296_B:1396_B:1496_B:1596_B:1696_B:1796_B:1896_B:1996_B:2096_B:2196_B:2296_B:2396_B:2496_B:2596_B:2696_B:2796_B:2896_B:29
c:hplcpreptamara96wellf.110prokchp2.gdt : WL_1 : P-504062-001X: Inj. Number: 1
c:hplcpreptamara96wellf.110prokchp2.gdt : WL_2 : P-504062-001X: Inj. Number: 1
Aqueous
Organic
* *
*
Wild type C. albicans hetEF31 – PSDVB Reverse Phase
2 – Ion Exchange
3 – RP HPLC
BioassayChromatographic Separation
Example - Yefafungin Bioassay-Guided Isolation
Roemer, et al., Chem Biol. 18 (2011) 148-164G,Harris ASP CS Workshop, July 21, 2018
11

12. Yefafungin
HN
N
O
O
O
OP
OCH3
O
H
NP
O
OH
O
H
NO
O
HO OH
Roemer, et al., Chem Biol. 18 (2011) 148-164
G,Harris ASP CS Workshop, July 21, 2018
12

13. 504094-
001G
0
50
100
-500
0
500
m
Vol
ts
0 20 40
Minute
s
Zero
F:1 F:2 F:3 F:4 F:5 F:6 F:7 F:8 F:9
F:10 F:11 F:12 F:13 F:14 F:15 F:16 F:17 F:18 F:19 F:20
Example - Colletofungins Bioactivity-Guided Isolation
0
100
200
300
400
500
600
0 20 40 60 80
L-472723
L-472724
min0 5 10 15 20 25 30
mAU
0
250
500
750
1000
1250
1500
1750
DAD1 C, Sig=210,16 Ref=360,100 (04_16_0204_16B.D)
0.338
1.068
2.031
2.388
3.1503.363
4.465
4.893
5.215
5.562
5.979
6.324
6.7316.914
7.319
7.962
8.394
8.862
9.244
9.631
10.042
10.441
10.676
10.98611.183
11.423
11.65711.847
12.117
12.47812.635
13.102
13.534
13.90714.09714.293
14.897
15.185
15.59315.81016.003
16.303
16.87017.04217.255
17.61417.825
18.069
18.854
19.32719.500
19.841
20.115
20.670
21.383
22.062
22.310
22.743
23.54023.749
25.591
26.419
26.927
27.890
DAD1 E, Sig=280,16 Ref=360,100 (04_16_0204_16B.D)
2.426
3.513
5.210
5.630
7.520
7.962
8.858
9.515
10.212
10.685
11.432
11.867
12.116
12.654
13.092
13.538
13.896
14.293
14.86515.027
15.544
16.272
16.69616.81417.000
17.529
18.279
18.507
18.85019.055
19.408
19.729
19.955
20.670
21.44121.61221.804
22.189
22.404
22.761
23.182
23.539
24.04424.243
24.714
25.118
25.746
26.132
26.836
27.151
PMP1, Solvent B
min0 5 10 15 20 25 30
mAU
0
500
1000
1500
2000
DAD1 C, Sig=210,16 Ref=360,100 (04_16_0204_16C.D)
1.225
1.6041.795
2.046
2.409
3.1543.366
4.8345.0445.221
5.848
6.268
6.804
7.0507.255
7.728
8.075
8.379
8.9529.166
9.523
10.047
10.331
10.702
10.98911.164
11.691
12.130
12.623
13.02513.161
13.548
13.91314.059
14.34514.53314.705
15.073
15.284
15.525
15.956
16.265
16.665
17.315
17.747
18.322
18.614
18.905
19.358
19.691
20.176
20.811
21.197
21.743
22.352
22.781
23.546
23.787
24.059
24.856
25.202
25.586
26.408
26.940
27.871
28.448
30.636
DAD1 E, Sig=280,16 Ref=360,100 (04_16_0204_16C.D)
2.432
3.427
8.189
8.656
8.894
9.521
10.435
10.712
10.95111.16111.366
11.708
12.135
12.636
12.867
13.238
13.57813.72613.922
14.34814.53414.722
15.019
15.288
15.504
15.856
16.07616.263
16.663
17.004
17.359
17.624
17.905
18.29218.493
19.198
19.691
20.354
20.795
21.447
21.797
22.493
22.791
23.166
23.542
24.016
24.234
24.723
25.108
25.830
26.049
26.296
26.673
27.918
28.440
29.059
PMP1, Solvent B
1 – PSDVB Reverse Phase
2 – CCC
3 – RP HPLC
G.Harris, et al. (Merck Research Labs) unpublishedG,Harris ASP CS Workshop, July 21, 2018
13

14. Colletofungins
CH3
OH
OH
OH
CH3
OH
O
CH3
CH3
CH3
CH3
CH3
O
CH3
OH
O
O
O
R
1
G.Harris, et al. (Merck Research Labs) unpublishedG,Harris ASP CS Workshop, July 21, 2018
14

15. Step 1 – Resolution and Recovery
• Recovery from biomatrix
– Method choice depends upon extraction solvent
• Water miscible or immiscible
– Commonly used methodologies
• Solid phase adsorption (SPE)
• Liquid-liquid partition
• CS
• Resolution of multiple biologically active components
– Low resolution methods sufficient
– Must be chemically “gentle”
– Reproducible
• pH control
G,Harris ASP CS Workshop, July 21, 2018
15

16. Elution-Extrusion (EECCC)
0 10 20 30 40 50
elution extrusion
Kd=1
Kd=0.01
Kd=0.2
Kd=5
Kd=100
System Parameters
CCC
Vc=135mL
F=6mL/min
Sf=0.8
N=1000
Time(min)
Berthod, A., Ruiz-Angel, M.J., Carda-Broch, S., Anal. Chem. 75 (2003), 5886-5894
Berthod, A., Hassoun, M. Harris, G.H., J. Liq. Chrom. Rel. Tech. 28 (2005), 1851-1866
Berthod, A., Friesen, B., Inui, T., Pauli, G. Anal. Chem. 79 (2007), 3371-3382
Kd=1
logKd=0
Exploiting the “support-free liquid stationary phase”
G,Harris ASP CS Workshop, July 21, 2018
16

17. 0.01
0.2
1
5
100
MP
SP
0.01
0.2
1 5
Berthod, A., Ruiz-Angel, M.J., Carda-Broch, S., Anal. Chem. 75 (2003), 5886-5894
Berthod, A., Hassoun, M. Harris, G.H., J. Liq. Chrom. Rel. Tech. 28 (2005), 1851-1866
Berthod, A., Friesen, B., Inui, T., Pauli, G. Anal. Chem. 79 (2007), 3371-3382
Classical
mode
Elution-Extrusion
MP
SP
after one
column volume
of SP
after SP
extrusion
Elution – Extrusion (EECCC)
No band broadening of SP
retained components
0.01
0.2 1
5
100
G,Harris ASP CS Workshop, July 21, 2018 17

18. Distribution of Example Plant Natural Products In
Standardized Solvent System
Armbruster, et al. J. Liq. Chrom. & Rel. Tech., 24 (2001) 1827-1840
Kd
Stationary PhaseMobile Phase
1
0.1
10
100
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49
LogKd
CCC Fraction Number
Glycyrhizzic Acid
Glycyrrhetinic Acid
Secologanin
Stigmasterol
Berberine
Brucine
Sparteine
Sanguinarine
Catechin
Umbelliferone
Esculetin
Genistein
Ellagic Acid
fatty
acids
polyphenolics
Kd=1
SS = CH2Cl2 : CH3OH : H2O (5:6:4); LP=MP
G,Harris ASP CS Workshop, July 21, 2018
18

19. HSCCC Fraction Number
Weight(mg)
200
180
160
140
120
100
80
60
40
20
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Weight Distribution of the 64 Fraction Sets
nonpolar polar
CH2Cl2 xt aqueous xt
“dual-mode” = EECCC
CH2Cl2 : CH3OH : H2O (5:6:4)
500 mg MeOH extract (hexane partitioned,
PVP treated)
Armbruster, et al. J. Liq. Chrom. & Rel. Tech., 24 (2001) 1827-1840G,Harris ASP CS Workshop, July 21, 2018
19

20. Generic Strategy for Purification of Crude Reaction Mixtures
Removal of Triphenylphosphine Oxide from Mitsunobu Reaction
Products – Purification of Reaction Products
• Purification of synthetic products from crude reaction mixtures containing
triphenylphosphine oxide can be problematic using typical separation methods
• A standard CCC strategy was developed based upon the one column volume
elution – extrusion methodology
• TPPO elutes at D=1 using HEMWat SS16
• Products more polar than TPPO elute early earlier; products more non-
polar than TPPO elute in column extrusion
• Advantages:
• Directly applicable to crude reaction mixtures – no sample preparation
• No method development for CCC
• Recovery and purity of products equivalent to that obtained using
preparative RP HPLC
• Complete recovery of entire reaction mixture
• TPPO retention independent of solvent system pH
Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56
G,Harris ASP CS Workshop, July 21, 2018
20

21. Mitsunobu Reaction Product Purification – Method and Theory
0 10 20 30 40
Time (min)
Absorbance254nm
1
2
0
1
2
3
4
5
6
0 0.5 1 1.5 2
intensity
Column Volumes
elution extrusion
A
B D=0.01
D=0.2
D=1
D=5
D=100
elution extrusion
Dynamic Extractions Spectrum HPCCC
Semi-preparative Column, Vc=135mL
1600rpm, 30 oC
Hexanes:EtOAc:MeOH:Water (5:6:5:6, v:v:v:v)
Reverse Phase Mode (upper phase stationary)
Stationary Phase Retention = 80%
Elution, LP @ F=6mL/min, 0 - 30min
Extrusion UP @ F=10mL/min, 30.1 – 46min
TPPO
tr~23’, D=1.02
Method Theory
polar non-polar
Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56
G,Harris ASP CS Workshop, July 21, 2018
21

22. Mitsunobu Reaction Product Purification – Example 5
0 10 20 30 40
Time (min)
Absorbance254nm
*
*
D=9.00
* TPPO
Dynamic Extractions Spectrum HPCCC
Semi-preparative Column, Vc=135mL
1600rpm, 30 oC
Hex:EtOAc:MeOH:Water (5:6:5:6, v:v:v:v)
Reverse Phase Mode (upper phase
stationary)
Stationary Phase Retention = 80%
Elution, LP @ F=6mL/min, 0 - 30min
Extrusion UP @ F=10mL/min, 30.1 – 46min
Crude Sample 229mg @
48% HPLC area purity
Pure Product 26.5mg @
>99% HPLC area purity
HPLC
HPCCC HPLC yielded 9mg product
@ 93% HPLC area purity
Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56
G,Harris ASP CS Workshop, July 21, 2018
22

23. Mitsunobu Reaction Product Purification – Example 11
0 10 20 30 40
Time (min)
Absorbance254nm
*
*
D=0.49
* TPPO
Dynamic Extractions Spectrum HPCCC
Semi-preparative Column, Vc=135mL
1600rpm, 30 oC
Hex:EtOAc:MeOH:Water (5:6:5:6, v:v:v:v)
Reverse Phase Mode (upper phase
stationary)
Stationary Phase Retention = 80%
Elution, LP @ F=6mL/min, 0 - 30min
Extrusion UP @ F=10mL/min, 30.1 – 46min
Crude Sample 182mg @
51% HPLC area purity
Pure Product 24.1mg @
92% HPLC area purity
HPLC yielded 5mg product
@ >99% HPLC area purity
HPLC
HPCCC
Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56
G,Harris ASP CS Workshop, July 21, 2018
23

24. Mitsunobu Reaction Product Purification – Example 10
0 10 20 30 40
Time (min)
Absorbance254nm
*
*
* TPPO
D=1.27
Crude Sample 219mg @
31% HPLC area purity
Pure Product 17.6mg @
98% HPLC area purity
HPLC yielded 18mg product
@ >99% HPLC area purity
Dynamic Extractions Spectrum HPCCC
Semi-preparative Column, Vc=135mL
1600rpm, 30 oC
Hex:EtOAc:MeOH:Water (5:6:5:6, v:v:v:v)
Reverse Phase Mode (upper phase
stationary)
Stationary Phase Retention = 80%
Elution, LP @ F=6mL/min, 0 - 30min
Extrusion UP @ F=10mL/min, 30.1 – 46min
HPLC
HPCCC
Edwards, N.A., et al., J.Chrom. A, 1323 (2014) 49-56
G,Harris ASP CS Workshop, July 21, 2018 24

25. Step 2 – Purification
• Chemical resolution of desired bioactive component(s) from inactive
components of extract
• Wide choice of methodology
– Moderate resolution sufficient if orthogonal
– Ideally one separation but can be a combination of two
– Common methods
• Normal phase chromatography
• Ion exchange
• CS
G,Harris ASP CS Workshop, July 21, 2018
25

26. 0
5
10
15
20
25
30
35
0 5 10 15 20
Time (min)
Adsorbance
0
5
10
15
20
25
30
35
0 5 10 15 20
Time (min)
Adsorbance
A
B
C
D
E
F
G
H
C
D
E
0
5
10
15
20
25
30
35
0 5 10 15 20
Time (min)
Adsorbance
C
D
E
Non-Orthogonal Separations
RP HPLC - A
RPHPLC-B
More or less
equivalent
separation
mechanisms
Harris, G.H., in The Handbook of Industrial Mycology, ed. Z. An, Marcel Dekker, New York, 2004
G,Harris ASP CS Workshop, July 21, 2018
26

27. 0
5
10
15
20
25
30
35
0 5 10 15 20
Time (min)
Adsorbance
0
5
10
15
20
25
30
35
0 5 10 15 20
Fraction (min)
Adsorbance
0
5
10
15
20
25
30
35
0 5 10 15 20
Time (min)
Adsorbance
A
B
C
D
E
F
G
H
CC
E
B
A
Orthogonal Separations
Polymeric RP
CCC(NP)
Reverse phase
adsorption followed by
normal phase partition
Harris, G.H., in The Handbook of Industrial Mycology, ed. Z. An, Marcel Dekker, New York, 2004
G,Harris ASP CS Workshop, July 21, 2018
27

28. -200
0
200
400
600
800
1000
1200
1400
0 5 10 15 20 25 30 35 40 45 50
Time (min.)
AreaCounts
MEK extract
EECCC Rich Cut, 2.6 mg
Example - Discovery Isolation of Moriniafungin
MEK extract
(40mg)
EECCC
solvent system: hexane:EtOAc:MeOH:H2O (5:5:5:5);UP=MP @ 3.0 mL/min., tail -> head
Conway Centrichrome DP1000, Vc=175mL, 800rpm
Preparative RP HPLC
Moriniafungin (~1.5 mg)
CO2H
CHO
OO
OH
CH3O
O
O
O
HO2C
moriniafungin
novel sordarin analog
Basilio, A., et al. Bioorganic and Medicinal Chemistry 14 (2006), 560-566G,Harris ASP CS Workshop, July 21, 2018
28

29. EECCC Based Strategy for Antifungal Fermentation
Extract Analysis and Isolation
• Two-step process consisting of two moderate resolution but
orthogonal separation methods
1) CHP20P - reverse phase adsorption, gradient elution increases
generality
2) EECCC – normal phase liquid-liquid partition, EECCC mode
increases generality
• Standardization yields chromatographic retention information useful
for dereplication and allows semi-automation
– Kd standardization allows for sample dependent variation of Sf
– LCMS analysis of fractions for early recognition of knowns
G,Harris ASP CS Workshop, July 21, 2018
29

30. EECCC Separation Conditions
• Instrumentation:
– Conway CentriChrome DP-1000 containing 2 – 175 mL bicoils (4
total coils)
– Control with in-house automated unit
• Operating Parameters:
– elution-extrusion mode, upper phase = mobile phase at
2.0mL/min (175mL total); extrusion with lower phase = stationary
phase (175.1mL to 367.5mL total elution volume); tail->head,
both processes at 800 RPM
– “Ito” type injection
– solvent system: hexane:EtOAc:MeOH:H2O (3:5:3:5)
– Sf routinely 0.75 – 0.85
An In-House Built Semiautomated Countercurrent Chromatography Workstation”, Napolitano, C., Wismer, M.,
Furlano, E., Harris, G., Uhrig, B., Blake, K., Kath, G., Dufresne, C., JALA, 2009, 27-35
G,Harris ASP CS Workshop, July 21, 2018
30

31. Hydrodynamic EECCC Separation of Antifungal
Standard Compounds
WF22210
pneumocandin Bo
enfumafungin
arundifungin
ascosteroside
extrusion
elution
Kd~1
200nm
CCC = Conway Centrichrome DP1000 (Vc=175mL)
N
O
NH
O
HO
NH
OH
HO
HO
O
N
OH
O
HN
OH
O
OH
NH
HO
O
NH
OH
H
H
H
O
H2N
O
N
O
NH
HO
O
H2N
O
NH
OH
HO
HO OSO3H
O
N
OH
O
HN
OH
O
OH
N
H
HOOOH
N
H
O
O
AcO
H
H
HO2C
OH
O
OH
HO
OH
HO
O
OH
OH
OH
OH
O
HO
O
O
OH
H
CO2H
O
OH
HO
CH3O
HO
G,Harris ASP CS Workshop, July 21, 2018
31

32. Hydrostatic EECCC Separation of Antifungal Standard
Compounds
extrusion
elution
Kd~1
WF22210
pneumocandin Bo
enfumafungin
arundifungin
ascosteroside
200nmCPC = Kromaton (Vc=1L)
N
O
NH
O
HO
NH
OH
HO
HO
O
N
OH
O
HN
OH
O
OH
NH
HO
O
NH
OH
H
H
H
O
H2N
O
N
O
NH
HO
O
H2N
O
NH
OH
HO
HO OSO3H
O
N
OH
O
HN
OH
O
OH
N
H
HOOOH
N
H
O
O
AcO
H
H
HO2C
OH
O
OH
HO
OH
HO
O
OH
OH
OH
OH
O
HO
O O
OH
H
CO2H
O
OH
HO
CH3O
HO
G,Harris ASP CS Workshop, July 21, 2018
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33. Distribution of Antifungal Activity in Routine Extracts
E-000002378
E-000002726
E-000900085
E-000504402
E-000504403
E-000504405
E-000504406
E-000504413
E-000504415
E-000504426
E-000504427
E-000504429
E-000504441
E-000504444
E-000504445
E-000504446
E-000504450
E-000504455
E-000504461
E-000504471
E-000504504
E-000504519
E-000504521
E-000504521
E-000504525
E-000504526
E-000504531
E-000504537
E-000504541
E-000504607
E-000504608
P-000502922
P-000502924
10 20 30
ascosteroside
ascosteroside analogs
unidenified triterpenes
arundifungin enfumafungin novel
papulacandins lipopeptides
Kd 0 0.5 1 1.5 2 4 8 16 90 ∞
fraction
polarity
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34. Step 3 – Polishing
• Final cleanup of desired compound
• Target compound identified
• Limited choice of methodology
– High resolution desired
– Common methodology
• Preparative RP HPLC
• Crystallization
• SFC
• CS
G,Harris ASP CS Workshop, July 21, 2018
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35. Example - Antifungal Cyclodepsipeptide Isolation
N
O
O
O
NHO
N
OCH3
O
NHO
NO
O
NH
N
N O
O
N
O
CO2H
J. Med. Chem. 1994, 37, 1908-1917
Discovery Isolation
Mitsubishi CHP20Y
Chromatography
Acetone Culture Extract
Standardized EECCC
H:E:M:W (3:5:3:5)
RP HPLC
Preparative Isolation
Kd < 0.1
Mitsubishi CHP20Y
Chromatography
Acetone Culture Extract
Optimized EECCC
H:E:M:W (5:4:5:4) Kd ~ 0.6
G,Harris ASP CS Workshop, July 21, 2018
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36. EECCC Solvent System Optimization for Cyclodepsipeptide
-100
100
300
500
700
900
1100
0 5 10 15 20
Time (min)
A280nm(AU*s)
3:5:3:5
5:5:5:5
5:4:5:4 5:3:5:3
extrusionelution MiniDE
Vc=5.4mL, 2200rpm
UP=MP, 0.5mL/min, T->H
Control using Agilent 1100 HPLC
0
500
1000
1500
2000
0 10 20 30 40
Fraction
mAU*s(280nm)
extrusionelution Conway CentriChrome “auto”
Vc=175mL, 800rpm
5:4:5:4, UP=MP, 3mL/min
9.67mL fractions (38)
Feed – CHP20P rich cut
74mg in 5mL UP + 5mL LP
for each of 2 coils
G,Harris ASP CS Workshop, July 21, 2018 36

37. Example - HSCCC Separation of Australifungin
Phenomenex Ultracarb 30 ODS, 9.4x250mm
55% CH3CN / 45% 25mM K2HPO4 pH6.8
55 oC, A280 nm
P.C. Inc. MLCPC, coil volume = 385 mL
hexane:EtOAc:MeOH: 25mM K2HPO4 pH6.8 (7:3:5:5), 3
mL/min, 800 rpm, T->H
A280 nm
- O.D. Hensens, et al. J. Org. Chem., 60 (1995) 1772-1776.
- S.M. Mandala, et al. J. of Antibiot., 48 (1995) 349-356.
Solid stationary phase interaction problems
G,Harris ASP CS Workshop, July 21, 2018 37

38. Example - CCC Separation of Viridiofungins
P.C., Inc. MLCPC, coil volume 385 mL, 800 rpm,
T->H, upper phase = MP,
hexane:EtOAc:MeOH:H2O+0.1%H3PO4 (5:5:5:5),
950 mg of crude tricarboxylic acid fraction obtained
from MEK extract
viridiofungin A (558 mg) recovered from extruded stationary phase
95
mg
30
mg
K=0.48 K=0.30
HO2C
HO CO2H
NHO
RHO2C
O OH
N
H
B R =
A R =
C R =
Viridiofungins: Harris, G.H., Jones, E.T.T., Meinz, M.S., Nallin-Omstead, M., Helms, G.L., Bills, G.F., Zink, D.,
Wilson, K.E. Tetr. Lett., 1993, 34, 5235-5238
Solvent System: Oka, F., Oka, H., Ito, Y, J. Chromatog. A., 1991, 538, 99-108
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39. Natural Product CS Literature
• There is a lot, many reviews for ideas,…..
• Is there a natural product that can’t be separated using CS?
• Focus on solvent system and mode of operation
– Useful recent review of elution mode operational modes: X.-Y.
Huang, Tr. Anal. Chem. (2016) 77, 214-225
• Useful recent compilations:
– Terpenes: K. Skalicka-Wozniak, I. Garrard, Phytochem. Rev.
(2014) 13, 547-572
– General: J.B. Friesen, et al., J. Nat. Prod. (2015) 78, 1765-1796
– General: K. Skalicka-Wozniak, I. Garrard, Nat. Prod. Rep. (2015)
32, 1556-1561
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40. Systematic Solvent System Selection
GUESS methodology
F.Oka, H. Oka, Y. Ito, J. Chrom. A (1991), 538, 99-108
Process Incorporation
Workflow section in latest CCS Review
Friesen et al., J. Nat. Prod. (2015), 78, 1765-1796
“Almost is good enough”
Elution – Extrusion CCC
Range of other CS operational modes available
G,Harris ASP CS Workshop, July 21, 2018
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41. Acknowledgements
Gilson Purification and Gregoire Audo
American Society of Pharmacognosy and Laura Stoll
G,Harris ASP CS Workshop, July 21, 2018 41