The document discusses various elution methods used in countercurrent chromatography, including elution-extrusion (EECCC), back-extrusion (BECCC), and intermittent countercurrent extraction (ICCE), highlighting their unique features and applications. It emphasizes the advantages of high-speed countercurrent chromatography, such as minimal sample preparation, high mass resolution, and reproducibility. The document also includes practical applications and examples of these methods in separating and purifying natural products.

1. Elution Methods

2. elution method phase changea
flow
direction
change
rotation
direction
change
comments
classical – – – highly retained analytes remain in stationary
phase
EECCC single – – analytes elute in order of K values
BECCC – single – elution order reverses; some analytes may
elute at separate volumes
back-step CCC twice – – a plug of aqueous phase introduced to elute
highly retained analytes
dual-mode single single – elution order reverses
dual-rotation single single elution order reverses
multiple dual-mode multiple multiple – elution order reverses each cycle
ICcE multiple multiple – sample loop is in the middle of a single
column or between two separate columns
recycling eluant fraction reintroduced into column
aphase change refers to switching mobile and stationary phase
Countercurrent Separation Elution Methods
Friesen JB, McAlpine JB, Chen SN, Pauli GF
Countercurrent Separation of Natural Products: An Update
Journal of Natural Products 78: 1765-1796 (2015)
dx.doi.org/10.1021/np501065h

3. Elution Methods: Classical

4. Reversibility of NP/RPA
280nm
230nm
A
K
0 0.125 0.25 0.375 0.5 0.625 0.75 0.875 1 1.14 1.33 1.6 2 2.67 4 8 ∞
IIII II
IIIII I1/K
GUESSmix in Hexane / Ethyl acetate / Methanol / Water 4:6:4:6
Reverse Phase
Normal Phase
G
H
X
T
r
C
D
F
R
U
V
A
Q
M
N
Z
E O
I
Y
b

5. Elution-Extrusion
http://images.wisegeek.com/grinder-extruding-clay.jpg

6. High Speed Countercurrent
Chromatography (HSCCC)
§Minimal sample preparation
(direct chromatography of crude extracts)
§High mass – High resolution
§No sample loss (support-free
chromatography)
§Reproducibility
(scale-up or
scale down)
§Flexibility

7. Dealing with wide ranges
of polarity
The CS answer to gradient
elution in LC
Elution-extrusion CCC

8. elution method phase changea
flow
direction
change
rotation
direction
change
comments
classical – – – highly retained analytes remain in stationary
phase
EECCC single – – analytes elute in order of K values
BECCC – single – elution order reverses; some analytes may
elute at separate volumes
back-step CCC twice – – a plug of aqueous phase introduced to elute
highly retained analytes
dual-mode single single – elution order reverses
dual-rotation single single elution order reverses
multiple dual-mode multiple multiple – elution order reverses each cycle
ICcE multiple multiple – sample loop is in the middle of a single
column or between two separate columns
aphase change refers to switching mobile and stationary phase
Summary of Countercurrent Separation Elution Methods: Elution-extrusion
Countercurrent Chromatography (EECCC), Back-extrusion Countercurrent
Chromatography (BECCC), and Intermittent Countercurrent Extraction (ICcE)
CCS Methods
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.

9. Elution-extrusion CCC
Fig. 2. The elution extrusion method.
(A) The elution step; (B) starting the
extrusion step by switching the
entering fluid; (C) the extrusion step.
A solvent front
moves through the column. (D and E)
Close view of the circled area
showing the difference in velocities
between the solvent front, uM, and the
“stationary” phase velocity, uS. The
dotted area in (D) is squeezed to fill
the volume in (E).
Band broadening inside the chromatographic column: The interest of a
liquid stationary phase Journal of Chromatography A, Volume 1126,
Issues 1–2, 8 September 2006, Pages 347-356 Alain Berthod

10. Fig. 3. Separation of five steroids compounds by elution–
extrusion CCC.
(1) Prednisone, (2) prednisolone acetate, (3) testosterone, (4) estrone, (5) cholesterol.
Liquid system: HepEMWat 5:6:5:6.
Aqueous lower phase
2 mL/min Sf = 0.6, 700 rpm.
Top: extrusion at 100 min (v = 200 mL);
middle: extrusion at 40 min (v = 80 mL);
bottom: extrusion at 25 min (v = 50 mL).
the hatched double arrows show the extrusion step
(machine volume VC = 52.2 mL). ELSD.
Band broadening inside the chromatographic column: The interest of a
liquid stationary phase Journal of Chromatography A, Volume 1126,
Issues 1–2, 8 September 2006, Pages 347-356 Alain Berthod
Elution-extrusion CCC

11. Elution-Extrusion CCC
Berthod, A.*; Friesen, J. B.*; Inui, T.; Pauli, G. F. [*equal contribution]
Elution-Extrusion Countercurrent Chromatography: Theory and Concepts in Metabolic Analysis.
Anal. Chem. 2007, 79, 3371-3382.

12. B
i
DVi
VC
VC·VCM · VRi
-1
h
g
C
g h
A
injected samples (g-l)
g h
VM VS
VCM·KDi
-1
l k j i
D
ihg jk l
E
V0
VCM
VCM+VM
VCM+VR(h)
Stage I
classical
elution
Stage II
sweep
elution
Stage III
extrusion
SDMR VKVV ii
×+=
elution equation
i
i
D
CM
CCMEECCC
K
V
VVV -+=
extrusion equation
new SP
solvent front
new
SP
VM·VCM · VRi
-1
il k j
Equilibrium - Start of EECCC Run
End of EECCC Run
chromatogram volumes
VCM+VC
N = 1 2 3 4 5 6 7 8 9 10
calculation
MP
MP SP
MP
SP
new SP
SP
SP
SP
Berthod, A. Friesen, J.B. Inui, T. Pauli G.F. Analytical Chemistry 79, 3371-3382 (2007)

13. 600m rpm 2 mL/min
0
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00
A254
600 rpm, 1.5 mL/min
0
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00
A254
600 rpm, 3 mL/min
0
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00
A254
Sf = 0.52
Sf = 0.47
Sf = 0.36
2.29 2.67 3.2 4.00 5.33 8.00 16
8
2.29 2.67 3.2 4.00 5.33 8.00 16
8
2.29 2.67 3.2 4.00 5.33 8.00 16
8
Increasing Flow Rate

14. GUESSmix in HEMWat 4:6:4:6
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'(2)
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 ¥K'(2)
A
280nm
230nm
J-type centrifuge 120 mL
Fast Centrifugal Partition Chromatography (FCPC) 200 mL
220 mg
440 mg
G
H
X
T
r
C
D
F
R
U
V
A
Q
M
N
Z
E O
I
Y
b
Instrument Comparison

15. HEMWat +3 VCM = 313 mL
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
HEMWat +3 VCM = 254.5 mL
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
HEMWat +3 VCM = 228 mL
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
HEMWat +3 VCM = 162 mL
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
I II III
a
b
c
d
MS

16. A
280nm
230nm
A
K
0 0.125 0.25 0.375 0.5 0.625 0.75 0.875 1 1.14 1.33 1.6 2 2.67 4 8 ∞
IIII II
IIIII I1/K
Reversed Phase
Normal Phase
G
H
X
T
r
C
D
F
R
U
V
A
Q
M
N
Z
E O
I
Y
b

17. KD
intervals
0
≤ KD < 0.0625
0.0625
≤ KD <
0.125
0.125
≤ KD < 0.25
0.25
≤ KD < 0.5
0.5
≤ KD <
1
1
≤ KD <
2
2
≤ KD <
4
4
≤ KD <
8
8
≤ KD <
16
16
≤ KD <
32
32
≤ KD <
∞
HEMWat
0 rXHTG DR CF
QUA
V
N ME Z O I Yb
DEMWat
0 rXHT G D C
FUV
A
RQ
ZMN
E
OI Yb
GUESS Mix in DEMWat 5:5:5:5
0 0.25 0.5 0.75 1 1.33 2 4 ∞K'(1)
A
HEMWat 0
0 0.25 0.5 0.75 1 1.33 2 4
∞K'(1)
A
280nm
230nm
O
I
Yb
Z
E
M
NA
VU
F
DRX
H
T
G
r
C
Q
I
X
H
T
r
G
D
C
F
U
V
A
R
Q
ZMNE
OIYb

18. EECCC applications
Fig. 2. “2VC” EECCC method for rapid screening of different HepEMWat liquid systems in reversed-phase separation mode.
CCC column of 140 mL. 3.0 mL/min, up to VCM (140 mL, 47 min, vertical dotted line) immediately followed by upper phase
flowing in the same direction; : 650 rpm: 20 mg crude extract dissolved in 2mL upper phase and 2mL lower phase.
Rapid and preparative separation of traditional Chinese medicine Evodia rutaecarpa employing elution-extrusion and back-extrusion counter-current chromatography: Comparative study
Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4140-4146 Yanbin Lu, Wenyan Ma, Ruilin Hu, Alain Berthod, Yuanjiang Pan

19. Fig. 3. “2VC” EECCC method for rapid screening of different Arizona liquid systems in normal-phase separation mode. (a)
Arizona liquid system N; (b) Arizona liquid system P. CCC column of 140 mL. Flow rate: 3.0 mL/min. up to VCM (140 mL, 47 min,
vertical dotted line) immediately followed by lower aqueous phase; 650 rpm; 20 mg crude extract in 2mL upper phase and 2mL
lower phase.
Rapid and preparative separation of traditional Chinese medicine Evodia rutaecarpa employing elution-extrusion and back-extrusion counter-current chromatography: Comparative study
Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4140-4146 Yanbin Lu, Wenyan Ma, Ruilin Hu, Alain Berthod, Yuanjiang Pan
EECCC applications

20. Separation and Purification Technology Volume 165, 2016, Pages 160–165
Computation-aided separation of seven components from Spirodela polyrrhiza (L.)
via counter-current chromatography Dabing Ren, Binsong Han, Zhongquan Xin,
Wenbin Liu, Shasha Ma, Yizeng Liang, Lunzhao Yi
Fig. 2. (A) EECCC separation results of ethyl acetate extract. Solvent system: HEEtWat
(1:3:1:3, v/v); stationary phase: upper phase. 2 mL/min: 900 rpm; 254 nm; temperature: 25 C;
Sf: 53.8%.
EECCC applications

21. Fig. 2. Schematic representation of elution–extrusion CCC methods. (A) Injection after equilibrium; (B) injection
before equilibrium; (C) sweep elution and extrusion at equilibrium. Eq., dynamic equilibrium of two phase. (I),
elution; (II), sweep elution; (III), extrusion. Red arrow: the point to inject the sample and pump simultaneously
mobile phase. Blue arrow: the point to switch pumped solvent from mobile phase to stationary phase. The white
bar (below the each graph): pumping mobile phase into the CCC column. The blue bar: pumping stationary phase
into the column.
J Chromatogr A. 2012 Feb 3;1223:53-63. doi: 10.1016/j.chroma.2011.12.036. Overlapping elution-extrusion counter-current chromatography: a novel
method for efficient purification of natural cytotoxic andrographolides from Andrographis paniculata. Wu D, Cao X, Wu S.
EECCC applications

22. J Chromatogr A. 2012 Feb 3;1223:53-63. doi: 10.1016/j.chroma.2011.12.036. Overlapping elution-extrusion counter-current chromatography: a novel
method for efficient purification of natural cytotoxic andrographolides from Andrographis paniculata. Wu D, Cao X, Wu S.
Fig. 4. ethanol extracts of A. paniculata. (A) Standard elution–extrusion
CCC method (tCM = 140 m) repeated elution–extrusion in) and (BCCC
(tCM,1 = 140 min and tCM,2 = 415 min, tj,2 = 275 min); (C) the
overlapping elution–extrusion CCC (tCM,1 = 85 min and tCM,2 = 250
min, tj,2 = 165 min). Peak (1) corresponding to andrographolide (1) and
peak (2,3) corresponding 14-deoxy-andrographolide (2) and 14-deoxy-
11,12-didehydroandrographolide (3). Other conditions: injection mode:
injection before equilibrium; elution mode: head-to-tail; 2 mL/min;: 850
rpm; :30 ◦C; 234.3 mg; UV detection: 254 nm; VS = 160 mL and VM =
110 mL; HEMWat 5:5:4:6 was prepared using an on-demand
preparation mode,. (I), elution; (II), sweep elution; (III), extrusion. Red
arrow: the point to inject the sample and pump simultaneously mobile
phase. Blue arrow: the point to switch pumped solvent from mobile
phase to stationary phase. Red dashed arrow: without third injection of
sample. The white bar (below the each graph): the pumped solvent
phase is lower phase used as mobile phase. The blue bar: the pumped
solvent phase is upper phase used as stationary phase.
EECCC
applications

23. Elution Methods: BECCC

24. Fig. 2. Separation of the test mixture in different configurations. (a) EECCC in the reversed-phase mode; (b) EECCC in the normal-phase mode; (c) back extrusion in the
reversed-phase mode; and (d) back extrusion in the normal-phase mode. VCM = 224 mL. The colored bands correspond to the liquid phases collected at the CCC column outlet.
The X-axis shows the elution volume in mL and the corresponding KD distribution coefficient expressed as [conc. in organic phase]/[conc. in aq. phase]. Injected amounts in
2mL mobile phase:
1-catechol (12 mg); 2-benzoic acid (8 mg); 3-benzaldehyde (2 mg); 4-anisole (20 mg); and 5-cumene (17 mg).
Using the liquid nature of the stationary phase in counter-current chromatography: V. The back-extrusion method
Journal of Chromatography A, Volume 1189, Issues 1–2, 2 May 2008, Pages 10-18 Yanbin Lu, Yuanjiang Pan, Alain Berthod
Elution Methods: BECCC

25. Fig. 3. Fractionation of an ethanol extract of Piper longum L. (b) BECCC with VCM = 140 mL. (c) EECCC with VCM = 140 mL. (d) BECCC with VCM = 350 mL.
Liquid system: HEMWat 3/2/3/2, aqueous mobile phase: 2.9 mL/min; VC = 140 mL; rotor rotation: 650 rpm; VM =93mL; VS =47mL; Sf = 34%; UV detection:
254 nm. Sample injection: 50 mg of dry extract dissolved in 1mL upper organic phase + 1mL lower aqueous phase. See Fig. 2 legend.
Using the liquid nature of the stationary phase in counter-current chromatography: V. The back-extrusion method
Journal of Chromatography A, Volume 1189, Issues 1–2, 2 May 2008, Pages 10-18 Yanbin Lu, Yuanjiang Pan, Alain Berthod
Elution Methods: BECCC

26. Fig. 3. Rapid extrusion CCC separation of E. rutaecarpa extracts using a
140mL CCC column with Arizona system Q in reversed-phase mode. (a)
EECCC. Flow rate: 3.0 mL/min of lower aqueous phase up to VCM (140 mL,
46min, vertical dotted line) immediately followed by 3 mL/min of upper
phase; post-column addition begins at 140 mL. (b) BECCC. Flow rate: 3.0
mL/min of lower aqueous phase. Valve switching at VCM (140 mL, 46min,
vertical dotted line); revolution speed: 650 rpm; detection: 254 nm;
injected sample: 100mg of crude extract in 2mL upper phase and 2mL
lower phase. (c) HPLC analysis of the extrusion CCC peak fractions I–V.
Rapid screening of bioactive components from Zingiber cassumunar using elution-extrusion counter-current chromatographyJournal of Chromatography A, Volume
1181, Issues 1–2, 15 February 2008, Pages 33-44 Yanbin Lu, Rui Liu, Alain Berthod, Yuanjiang Pan
Elution Methods: BECCC

27. Fig. 3. HSCCC separation of pigments extracted from spinach. (N) Neoxanthin, (V) violaxanthin, (L) lutein, (C) -carotene,
(Chl a) chlorophyll a and (Chl b) chlorophyll b. The arrow indicates the stop of rotation (295 min) to elute the less polar
compounds in reversed mode (tail to head). Conditions: solvent system: HEtWat (6:5:1.3); mobile phase: lower phase;
flow rate: 1.0 ml/min; revolution speed: 1050 rpm; sample size: 200 mg.
Journal of Chromatography A Volume 1074, Issues 1–2, 13 May 2005, Pages 99–105
Isolation of carotenoids from plant materials and dietary supplements by high-speed
counter-current chromatography Robert Aman, Reinhold Carle, Jürgen Conrad, Uwe
Beifuss, Andreas Schieber ,
Elution Methods: BECCC

28. Elution Methods: Back-Step

29. Elution Methods: Dual-Mode

30. Berthod2007_JLCRT_30_1447_LoveStory
Elution Methods: Dual-Mode

31. Elution Methods: Dual-Rotation

32. Elution Methods:
Multiple Dual Mode

33. head
tail
mobile
stationary
mobile
stationary
mobile
stationary
mobile
stationary
mobile
stationary
upper
lower
in out
Elution Methods:
Multiple Dual Mode
Friesen JB, McAlpine JB, Chen SN, Pauli GF
Countercurrent Separation of Natural Products: An Update
Journal of Natural Products 78: 1765-1796 (2015)
dx.doi.org/10.1021/np501065h

34. Fig. 3. Diagram of the multiple dual mode set-up in the head-to-tail or descending position (A) and tail-to-head or
ascending position (B). Solute 1 elutes immediately in the descending step, and solutes 4 and 5 with a high affinity
for the upper phase elute in the second ascending step, while the remaining solutes 2 and 3 see increased separation
going back and forth in the following dual mode steps.
Purification of Coomassie Brilliant Blue G-250 by multiple dual mode countercurrent chromatography Journal of Chromatography A, Volume 1232, 6 April 2012, Pages 134-141
Nazim Mekaoui, Joseph Chamieh, Vincent Dugas, Claire Demesmay, Alain Berthod
Multiple Dual Mode

35. Using the liquid nature of the stationary phase. VI. Theoretical study of multi-dual mode countercurrent chromatography Journal of Chromatography A,
Volume 1218, Issue 36, 9 September 2011, Pages 6061-6071 Nazim Mekaoui, Alain Berthod
Fig. 6. Experimental separation of dinitrophenyl derivatives of alanine (first peak) and glutamine (second peak). (A) Classical separation with a 15 mL
hydrostatic CCC column and the HEM/aqueous HCl 0.1 M; 1:1:1:1, lower aqueous mobile phase in the descending or head-to- tail direction at 0.3
mL/min, VS = 5.1 mL, VM = 9.9 mL, 1000 rpm, detection UV 280 nm, KD1 = 0.42, KD2 = 1.18. (B) MDM mode with 29 steps as indicated performed with
a constant flow rate of 0.3 mL/min for both liquid phases. (C) MDM mode with 14 steps of double volume and also constant flow of 0.3 mL/min. (D)
Classical elution at 0.1 mL/min.
Multiple Dual Mode

36. Fig. 7. Experimental separation of dinitrophenyl derivatives of serine (first peak) and aspartic acid (second peak). (A) Classical separation with a 130 mL hydrodynamic CCC column
and HEM/aqueous HCl 0.1 M; 4:5:4:5, lower aqueous mobile phase in the descending or head-to-tail direction at 0.35 mL/min, VS = 100 mL, VM = 30 mL, 800 rpm, detection UV
280 nm, KD1 = 0.77, KD2 = 0.90. (B) Classical separation but with mobile phase flow rate 2 mL/min, VS = 74 mL, VM = 56 mL; (C) MDM mode with 67 steps as indicated performed
with a constant flow rate of 2 mL/min for both liquid phases. (D) MDM mode with 33 steps of double volume and also constant flow of 2 mL/min.
Using the liquid nature of the stationary phase. VI. Theoretical study of multi-dual mode countercurrent chromatography Journal of Chromatography A,
Volume 1218, Issue 36, 9 September 2011, Pages 6061-6071 Nazim Mekaoui, Alain Berthod
Multiple Dual Mode

37. Fig. 4. Separation of polar and non-polar compounds
in Coomassie Blue G-250 (Acros #1) by dual-mode
elution and control by TLC. CCC conditions:
HepBuWat 2:3:4 system. 1000 rpm; both H → T
descending and T→ H ascending flow rates: 2
mL/min; Sf = 46%; injection volume: 1 mL (10 mg);
classical descending CM step for 38.5 min or 77 mL
aqueous phase (wavy blue band); dual mode DM step
until complete elution of the hydrophobic fraction at
78.5 min after 80 mL organic phase (dotted red band).
TLC conditions: silica gel on aluminum Plates 60
F254, 1-butanol/acetic acid/water 75:10:5 (v/v) eluting
phase. The TLC controlled fractions are indicated.
Purification of Coomassie Brilliant Blue G-250 by multiple dual mode countercurrent chromatography Journal of Chromatography A, Volume 1232, 6 April 2012, Pages 134-141
Nazim Mekaoui, Joseph Chamieh, Vincent Dugas, Claire Demesmay, Alain Berthod
Multiple Dual Mode

38. Fig. 5. 254 nm UV trace obtained during a “trapping” multi-dual-mode experiment for the purification of 500 mg of CBB G-250 (Acros #1).
Two coils serially connected (see Fig. 3 for experimental set-up). Total volume 140 mL. Rotor rotation: 1100 rpm; flow rate (both phases): 2
mL/min; Sf = 46%. Blue steps refer to head-to-tail descending elution step with the aqueous phase and orange to tail-to-head ascending step
with the organic upper phase. Repetitive 1 mL injections are indicated by arrows. The light yellow bands show the polar fraction elution (almost
colorless) collected in the aqueous phase. The pink bands shows the apolar blue fractions collected in the organic phase. The purified CBB
fraction was recovered in a long 70 mL head-to-tail aqueous phase elution after 155 min not shown due to complete detector saturation. See
Table 1 for full modeling of band locations inside the CCC columns.
Purification of Coomassie Brilliant Blue G-250 by multiple dual mode countercurrent chromatography Journal of Chromatography A, Volume 1232, 6 April 2012, Pages 134-141
Nazim Mekaoui, Joseph Chamieh, Vincent Dugas, Claire Demesmay, Alain Berthod
Multiple Dual Mode

39. Elution Methods: ICcE
Intermittent Countercurrent Extraction

40. Intermittent counter-current extraction as an alternative approach to purification of Chinese herbal
medicine Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4187-4192 Peter
Hewitson, Svetlana Ignatova, Haoyu Ye, Lijuan Chen, Ian Sutherland
Fig. 1. ICcE operating modes. Valves V1 and V2 were switched to allow operation in either
normal phase (upper phase mobile) or reversed phase (lower phase mobile).
Elution Methods: ICcE

41. Fig. 2. (a) UV chromatogram and (b) chromatogram
constructed from HPLC peak areas using an
intermittent counter-current extraction method on
a Midi-DE preparative column for the separation of
four compounds from the GUESSmix (Caffeine (C),
K = 0.09; Vanillin (V), K = 0.55; Naringenin (N), K =
1.25 and Carvone (O), K = 7.4).
Solvent system: HEMWat-16a; upper phase flow
rate 35 ml/min; lower phase flow rate 40 ml/min;
flow switched every 4min; sample concentration:
28.4 g/l, sample
volume: 40 ml; rotational speed: 1250 rpm; upper
phase detection wavelength: 230 nm; lower phase
detection wavelength: 273 nm temperature: 30 ◦C.
Intermittent counter-current extraction as an alternative approach to purification of Chinese herbal
medicine Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4187-4192 Peter
Hewitson, Svetlana Ignatova, Haoyu Ye, Lijuan Chen, Ian Sutherland

42. Fig. 4. Quasi-continuous counter-current-
chromatography scale-up from(a)912mL
Midi to (b) 4.6 L Maxi.
Operating conditions for Midi [14]: speed,
1250 rpm, upper and lower phase flow, 60
mL/min, time interval, 4 min; sample load
11.2 g total in 14 min.
Operating conditions for Maxi: speed, 600
rpm, upper and lower phase flow, 250
mL/min; sample loading 40.5 g in 20 min.
Phase system: HEMWat (4:5:4:5).
K values: caffeine (0.09); vanillin (0.55);
naringenin (1.25) and carvone (7.39).
Scale-up of counter-current chromatography: Demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale
Journal of Chromatography A, Volume 1216, Issue 50, 11 December 2009, Pages 8787-8792 Ian Sutherland, Peter Hewitson, Svetlana Ignatova

43. Fig. 4. Chromatogram constructed from HPLC peak areas using an ICcE method
on a Midi-DE preparative column for the extraction of tritolide from a dried
down MPLC fraction from an ethanol extract of Tripterygium Wilfordii Hook. f.
(bioactive components – Triptolide (C1), K = 1.07; Peritassines A (C2), K = 2.90;
wilforigine (C3), K = 10.2 and wilforine (C4), K = 13.8); Solvent system:
HEMWat 15; upper phase flow rate 40ml/min; lower phase flow rate 35 ml/min;
flow switched every 4min; sample concentration: 12.0 g/l, sample volume:
766ml; rotational speed: 1250 rpm; upper phase detection wavelength: 226 nm;
lower phase detection wavelength: 220 nm; temperature: 30 ◦C.
188mg of triptolide at greater than
98% purity was separated from 9.2
g of crude extract, using 10 l of
solvent in
3h.
Intermittent counter-current extraction as an alternative approach to purification of Chinese herbal
medicine Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4187-4192 Peter
Hewitson, Svetlana Ignatova, Haoyu Ye, Lijuan Chen, Ian Sutherland

44. Elution Methods: Dual Flow

45. Evaluation of dual flow counter-current chromatography and intermittent counter-current extraction Journal of Chromatography A, Volume 1218, Issue
36, 9 September 2011, Pages 6102-6106 Svetlana Ignatova, Peter Hewitson, Ben Mathews, Ian Sutherland
Fig. 5. Fractogram constructed from HPLC fraction analysis
after DFCCC separation of four compounds from the
GUESSmix (caffeine (C), K = 0.14; vanillin (V), K = 1.21;
naringenin (N), K = 3.82 and carvone (O), K = 14.8). Solvent
system: HEMWat 15;
upper phase and lower phase flow rate 35 ml/min;
sample concentration: 50.0 g/l,
sample volume: 150 ml;
rotational speed: 1000 rpm;
temperature: 30 ◦C.
Elution Methods: Dual Flow

46. Evaluation of dual flow counter-current chromatography and intermittent counter-current extraction Journal of Chromatography A, Volume 1218, Issue
36, 9 September 2011, Pages 6102-6106 Svetlana Ignatova, Peter Hewitson, Ben Mathews, Ian Sutherland
Elution Methods: Dual Flow

47. Elution Methods: Cocurrent

48. Fig. 5. Actual chromatogram of the separation by cocurrent CCC of
five steroids of Table 1. Liquid system: HepEMWat 5:6:5:6. Mobile
phase: lower aqueous phase, flow rate 2 mL/min; slower phase: upper
phase at 0.5 mL/min flow rate. Machine volume VT = 53 mL. 800 rpm.
Detection ELSD. Peak order: (1) prednisone (0.32 mg), (2)
prednisolone acetate (0.34 mg), (3) testosterone (0.42 mg), (4) estrone
(1.5 mg) and (5) cholesterol (1.1 mg). Injection volume 200 L of the
steroids in lower phase.
Band broadening inside the chromatographic column: The interest of a liquid stationary phase
Journal of Chromatography A, Volume 1126, Issues 1–2, 8 September 2006, Pages 347-356
Alain Berthod
Elution Methods: Cocurrent

49. Anal Bioanal Chem. 2015 Dec;407(30):9019-28. doi:
10.1007/s00216-015-9068-5.
Accelerated separation of GC-amenable lipid classes in plant
oils by countercurrent chromatography in the co-current
mode. Hammann S, Englert M, Müller M, Vetter W
Elution Methods: Cocurrent

50. Cocurrent
Fig. 3 CCC-ELSD chromatogram of a) the
separation of the standard
mixture and b) elution of the standard
compounds, determined by offline
GC/MS analysis of CCC fractions after
trimethylsilylation
H/benzotrifluoride/Ac 100:35:65 Fs = 4 mL/min
Fm = 2 mL/min UP mobile
Anal Bioanal Chem. 2015 Dec;407(30):9019-28. doi: 10.1007/s00216-015-9068-5.
Accelerated separation of GC-amenable lipid classes in plant oils by countercurrent chromatography in the co-current mode.
Hammann S, Englert M, Müller M, Vetter W

51. HSCCC chromatograms of the EPS with (A)OPB mode and (B) DPB mode. HSCCC conditions: solvent system: EBuWat (9:1:10); : 900
rpm; : 30 °C; flow rate: 1.8mL/min; detection wavelength: 280 nm; sample size: (A) 200mg of the EPS in 10 mL of the lower phase; (B)
50mg of the EPS in 5mL of the upper phase and 5mL of the lower phase.
In the HSCCC separation procedure, the two phases of the solvent
system EBuWat (9:1:10) were pumped into the coil column at a flow rate of
20 mL/min with two constant flow pumps. After the column was entirely
filled with the solvent system and rotating at 900 rpm, the flow rate of both
the two phases was adjusted to 2.2 mL/min. Only the lower phase was eluted
out from the column in the equilibration process. Equilibrium was established
when the two phases eluted from the outlet of the column had the same
volume
Phytochem Anal. 2015 Nov-Dec;26(6):444-53. doi: 10.1002/pca.2579. Rapid Separation of Three Proanthocyanidin Dimers from Iris lactea Pall.
var. Chinensis (Fisch.) Koidz by High-Speed Counter-Current Chromatography With Continuous Sample Load and Double-Pump Balancing Mode.
Lv H, Yuan Z, Wang X, Wang Z, Suo Y, Wang H.

52. Elution Methods: Recycling

53. Recycling CCS
Journal of Chromatography A, 1127 (2006) 298–301 Preparative separation of gambogic acid and its C-2
epimer using recycling high-speed counter-current chromatography Quan Bin Han, Jing Zheng Song, Chun
Feng Qiao, Lina Wong, Hong Xi Xu∗
Fig. 3. Chromatogram of gambogic acids by preparative recycling
countercurrent chromatography. Solvent system: n-hexane–methanol–
water (5:4:1, v/v/v); stationary phase: upper organic phase; mobile
phase: lower aqueous phase;
flow-rate: 2.0 ml/min; revolution speed: 800 rpm; sample: 50 mg
dissolved in 5ml of lower phase.

54. Fig. 3. (A) Online recycling HSCCC separation of Fr I. Solvent system: HEEtWat (1:9:1:9); Sf:
59.2%. stationary phase: upper phase; flow rate: 2 mL/min; rotation speed: 900 rpm;
detection wavelength: 254 nm; temperature: 25 C.
Separation and Purification Technology Volume 165, 2016, Pages 160–165
Computation-aided separation of seven components from Spirodela polyrrhiza (L.)
via counter-current chromatography Dabing Ren, Binsong Han, Zhongquan Xin,
Wenbin Liu, Shasha Ma, Yizeng Liang, Lunzhao Yi
Recycling CCS

55. J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Sep 15;1001:82-9. doi: 10.1016/j.jchromb.2015.07.051. Separation of phenolic acids and flavonoids
from Trollius chinensis Bunge by high speed counter-current chromatography. Qin Y, Liang Y, Ren D, Qiu X, Li X.
Fig. 6. (A) HSCCC chromatogram of Fr1. Solvent system, HEMWat (1:9:1:9); Sf , 63.1%. (B) HSCCC chromatogram of Fr2. Solvent system, HEMWat
(1:4:1:4); Sf , 48.5%. (C) HSCCC chromatogram of Fr4 with the recycling elution mode. Solvent system, HEMWat (1:2:1:2); Sf , 61.5%. (D) HSCCC
chromatogram of Fr6. Solvent system, HEMWat (1:2:1:2); Sf , 61.5%. A–D possess the same operation conditions: stationary phase, upper phase;
flow rate, 2 mL/min; rotation speed, 900 rpm; temperature, 25 ◦C.
Recycling CCS