What are the different parameters to consider for the modification of a chosen solvent system to be utilized in countercurrent chromatography.

1. CounterCurrent Separation
Theory and practice
§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
§Mild conditions for sensitive molecules

2. Modification of Solvent Systems
Phase modification before (column) pre-equilibration
- Inorganic salts
- DMSO / Ionic Liquids
- Acid and/or Base
- Ion-Pairing Factors
- Salts that Create Hydro-alcoholic Systems

3. Modification of Solvent Systems
Phase modification before (column) pre-equilibration
- Inorganic salts

4. addition of salts
Salting-out gradients in centrifugal partition chromatography for the isolation of chlorogenic acids from green coffee Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4245-4251
Roman R. Romero-González, Robert Verpoorte
Fig. 2. Salting effect of selected salts on chlorogenic acid, 5-CQA, in an ethyl acetate-aqueous salt solution (pH 2.5)
biphasic system. Distribution constants are defined as the
concentration ratio between the most lypophilic phase and the most hydrophilic one. Maximum salt concentrations
are determined by their solubility limits.

5. Addition of Salts
Partition efficiencies of newly fabricated universal high-speed counter-current chromatograph for
separation of two different types of sugar derivatives with organic-aqueous two-phase solvent systems
Kazufusa Shinomiya J Chromatogr A 1322:74-80. 2013

6. Partition efficiencies of newly fabricated universal high-speed counter-current chromatograph for
separation of two different types of sugar derivatives with organic-aqueous two-phase solvent systems
Kazufusa Shinomiya J Chromatogr A 1322:74-80. 2013
Addition of Salts

7. Anal Bioanal Chem. 2011 Jul;400(10):3615-23. doi: 10.1007/s00216-011-4995-2.
High-speed counter-current chromatographic separation of phytosterols.
Schröder M1, Vetter W.
Addition of Salts

8. Modification of Solvent Systems
Phase modification before (column) pre-equilibration
- DMSO / Ionic Liquids

9. DMSO additive
Figure 1. HPLC fractogram of a CPC purification of protected exenatide (2 g); solvent system:
Heptane/THF/MeCN/DMSO/W, 19.1:36.2:14:17.6:13 v/v; descending mode,flow rate: 4 mL/min plus 5%
stationary phase co-current; rotation speed: 2000 rpm; stationary phase retention: 74%; UV detection: =
215 nm; RRT: relative retention time in HPLC.
HepTetAcSoWat
J Chromatogr A. 2014 Apr 11;1337:155-61. doi: 10.1016/j.chroma.2014.02.052.
Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography.
Amarouche N, Giraud M, Forni L, Butte A, Edwards F, Borie N, Renault JH.

10. Methyl furan and N-methyl pyrrolidone
Figure 2. HPLC fractogram of a CPC purification of 8-mer protected peptide (1 g); solvent system:
Heptane/Me-THF/NMP/water, 7:50:23:20, v/v; descending mode, flowrate: 8 mL/min; extrusion step at
31 min, rotation speed: 1400 rpm; stationary phase retention: 72%; UV detection: = 215 nm; RRT: relative
retention time in HPLC.
Hep/Me-THF/NMP/Wat
J Chromatogr A. 2014 Apr 11;1337:155-61. doi: 10.1016/j.chroma.2014.02.052.
Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography.
Amarouche N, Giraud M, Forni L, Butte A, Edwards F, Borie N, Renault JH.

11. Modification of Solvent Systems
Phase modification before (column) pre-equilibration
- Acid and/or Base

12. Influence of pH on K
Fig. 1. Effect of the total benzoic acid concentration (top) and aqueous phase pH (bottom) on the benzoic acid distribution ratio, KC. Heptane to
buffer volume ratio is one to one. The inset in the top figure shows the linear change of the benzoic acid distribution ratio at pH 7. The inset in
the bottom figure shows the benzoic acid adsorption isotherms (concentration in the aqueous phase versus concentration in the heptane
phase) at different mobile phase pH values (1, 3, 4.2, 5 and 6).
Journal of Chromatography A, 1218 (2011) 6024–6030 Distribution ratio, distribution constant and partition coefficient. Countercurrent chromatography retention of benzoic acid
Alain Berthod∗, Nazim Mekaoui

13. Fig. 3. Retention of benzoic acid at different pH
values. Liquid system Composition 2,
heptane/butanol/buffer 200/20/380 (Table 2).
Mobile aqueous lower phase at 0.8 mL/min in
the descending head-to-tail direction.
Hydrodynamic CCC column Milli, 18 mL, rotor
rotation speed 2000 rpm, 30 ◦C. Benzoic acid
injected mass:
0.5 mg, injected volume: 0.5 mL. UV detection
at 254 nm. Aqueous buffer pHs are: A – 6.96; B
– 4.61; C – 3.62; and D – 2.19.
Journal of Chromatography A, 1218 (2011) 6024–6030 Distribution ratio, distribution constant and partition coefficient. Countercurrent chromatography retention of benzoic acid
Alain Berthod∗, Nazim Mekaoui
Influence of pH on K

14. Influence of pH on K
Countercurrent purification of the tetrahydro iso-α acids derived from Humulus lupulus L. JOURNAL OF SEPARATION SCIENCE
Volume 33, Issue 17-18, September 2010, Pages: 2828–2832, Clinton J. Dahlberg, James S. Traub, Matthew L. Tripp, Jeffrey S. Bland and Brian J. Carroll

15. Influence of pH on K
Countercurrent purification of the tetrahydro iso-α acids derived from Humulus lupulus L. JOURNAL OF SEPARATION SCIENCE
Volume 33, Issue 17-18, September 2010, Pages: 2828–2832, Clinton J. Dahlberg, James S. Traub, Matthew L. Tripp, Jeffrey S. Bland and Brian J. Carroll
Figure 2. Overlay of the UV
chromatogram from the CS
experiment (entry 3, Table 1, 254 nm)
with a reconstructed CS
chromatogram (254 nm) from the
HPLC analysis of collected
28 mL fractions.
HEMWat (acetate pH 5.3)
7:3:5:5

16. Influence of pH on K
Isolation of bitter acids from hops (Humulus lupulus L.) using countercurrent chromatography
JOURNAL OF SEPARATION SCIENCE Volume 35, Issue 9, May 2012, Pages: 1183–1189, Clinton J. Dahlberg, Guy Harris, Jan Urban, Matthew
L. Tripp, Jeffrey S. Bland and Brian J. Carroll
Figure 5. CCC chromatogram (254 nm) using a
HEMWat 5:5:5:5,
250 mM NH4Ac; pH 5.1 solvent system for the
purification of the RIAA diastereomers
produced by NaBH4 reduction of the C6
keto in cis n-IAA (isohumulone).

17. Modification of Solvent Systems
Phase modification before (column) pre-equilibration
- Ion-Pairing Factors

18. Ion Pairs
Fig. 2. HSCCC-chromatogram of C18-enriched pigment extract (1677 mg) I. lindeniiVan Houtte leaves.
HSCCC conditions: flow rate: 3.0 mL/min; ‘head-to-tail mode’; biphasic solvent system TBME–1-BuOH–
ACN–H2O (0.7% HFBA) 1:3:1:5 (v/v/v/v).Below, HPLC retention times of the separated major
betacyanins detected in each HSCCC fraction.
Separation of amaranthine-type betacyanins by ion-pair high-speed countercurrent
chromatography
Journal of Chromatography A, Volume 1344, 30 May 2014, Pages 42-50
Gerold Jerz, Nadine Gebers, Dominika Szot, Maciej Szaleniec, Peter Winterhalter,
Slawomir Wybraniec
https://nl.wikipedia.org/wiki/Betanine#/media/File:Betanin.svg

19. Separation of amaranthine-type betacyanins by ion-pair high-speed countercurrent chromatography
Journal of Chromatography A, Volume 1344, 30 May 2014, Pages 42-50
Gerold Jerz, Nadine Gebers, Dominika Szot, Maciej Szaleniec, Peter Winterhalter, Slawomir Wybraniec
Target-guided separation of Bougainvillea glabra betacyanins by direct coupling of preparative ion-pair
high-speed countercurrent chromatography and electrospray ionization mass-spectrometry Journal of
Chromatography A, Volume 1217, Issue 27, 2 July 2010, Pages 4544-4554 Gerold Jerz, Sławomir
Wybraniec, Nadine Gebers, Peter Winterhalter
terBuAcWat (HFBA) 1:3:1:5
terBuAcWat (TFA) 2:2:1:5
Separation of betalains from berries of Phytolacca americana by ion-pair high-speed counter-current
chromatography Journal of Chromatography A, Volume 1190, Issues 1–2, 9 May 2008, Pages 63-73 Gerold
Jerz, Tanja Skotzki, Kathrin Fiege, Peter Winterhalter, Sławomir Wybraniec
BuAcWat (TFA) 5:1:6 (0.7%)
Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed
countercurrent chromatography
Journal of Chromatography A, Volume 1216, Issue 41, 9 October 2009, Pages 6890-6899
Sławomir Wybraniec, Paweł Stalica, Gerold Jerz, Bettina Klose, Nadine Gebers, Peter Winterhalter, Aneta
Spórna, Maciej Szaleniec, Yosef Mizrahi
BuAcWat (TFA) 5:1:6 (0.7%)
terBuAcWat (TFA/HFBA) 2:2:1:5 (0.7%)
New solvent systems for gradient counter-current chromatography in separation of betanin and its
derivatives from processed Beta vulgaris L. juice.
Journal of Chromatography A, Volume 1380, 6 February 2015, Pages 29-37
Aneta Spórna-Kucab, Ian Garrard, Svetlana Ignatova, Sławomir Wybraniec
BuEtWat/Aq. NaCl/Aq. H3PO4
1300:200-1000:1300:700:2.5-10
Ion-pair high-speed countercurrent chromatography in fractionation of a high-molecular weight variation
of acyl-oligosaccharide linked betacyanins from purple bracts of Bougainvillea glabra
Journal of Chromatography B, Volume 878, Issues 5–6, 15 February 2010, Pages 538-550
Sławomir Wybraniec, Gerold Jerz, Nadine Gebers, Peter Winterhalter
BuAcWat (TFA) 5:1:6 (0.7%)
terBuAcWat (TFA) 2:2:1:5 (0.7%)
Fig. 3. IP–HSCCC chromatogram of directly recovered crude pigment extracts (755
mg) of violet B. glabra bracts in the solvent system terBuAcWat 2:2:1:5 using 0.7%
TFA. HSCCC conditions: flow rate: 3.0 mL/min; CCC-operation: head-to-tail mode;
velocity: 850 rpm and detection wavelength 540 nm.
Ion Pairs

20. Modification of Solvent Systems
Phase modification before (column) pre-equilibration
- Salts that Create Hydro-alcoholic Systems

21. Hydroalcoholic Systems
Journal of Liquid Chromatography & Related Technologies
Volume 29, Issue 5, 2006 Countercurrent Chromatographic Separation of Biotic Compounds with Extremely Hydrophilic Organic-Aqueous Two-Phase Solvent Systems and Organic-Aqueous
Three-Phase Solvent Systems
DOI:10.1080/10826070500509298 Kazufusa Shinomiya & Yoichiro Ito
Figure 1. CCC Chromatogram of proteins
obtained by the X-axis CPC using ethanol/
aqueous 2M ammonium sulfate (3 : 5) with the
upper phase mobile. Experimental
conditions: apparatus: X-axis CPC equipped
with a pair of eccentric coil assemblies with
1mm ID and a total capacity of 26.5 mL;
sample: 5mg each of human serum albumin
and lysozyme; solvent system:
ethanol/aqueous 2M ammonium sulfate (3 :
5); mobile phase: upper phase; flow rate:
0.4mL/min; revolution: 800 rpm. SF ¼ solvent
front.

22. Hydroalcoholic Systems
Counter-current chromatographic separation of nucleic acid constituents with a hydrophilic solvent system Journal of Chromatography A, Volume 1217, Issue
20, 14 May 2010, Pages 3457-3460 Yoichi Shibusawa, Atsushi Shoji, Chihiro Suzuka, Akio Yanagida, Yoichiro Ito
Fig. 3. Separation of eight nucleic acid constituents by HSCCC using hydrophilic solvent system. Dimension of PTFE multilayer coiled
column: 1.0mm I.D.×50m (40 ml capacity); solvent system: 1-propanol/800mM potassium phosphate buffer (pH 7.4) (1:1, v/v); stationary
phase: upper phase; mobile phase: lower phase; flowrate: 0.5 ml/min; revolution: 1000 rpm; UP = the starting point at which the upper
phase was used as the mobile phase. For the components’ identification, see Fig. 1.

23. Modification of Solvent Systems
Phase modification before (column) pre-equilibration
- Hydrophobic Compounds

24. Hydrophobic Compounds
Fig. 1. Partition coefficients of four critical pairs of fatty acids versus the percentage of water in the mobile phase in
HepMWat. 1 = C16:3; 2 = C18:4; 3 = C20:5; 4 = C22:6.
Counter-current chromatographic separation of polyunsaturated fatty acids
Journal of Chromatography A, Volume 704, Issue 1, 2 June 1995, Pages 211-216
Olivier Bousquet, François Le Goffic

25. Hydrophobic Compounds
(a) methyl p-hydroxybenzoate, (b) ethyl p-hydroxybenzoate, (c) ethyl m-hydroxybenzoate (d) isopropyl p-
hydroxybenzoate, (e) propylp-hydroxybenzoate, (f) isobutyl p-hydroxybenzoate and (g) butyl p-
hydroxybenzoate
HCyterMWat 5:0:2:5:3
HCyterMWat 4:1:2:5:3
HCyterMWat 3:2:2:5:3
HCyterMWat 2:3:2:5:3
HCyterMWat 1:4:2:5:3
HCyterMWat 0:5:2:5:3
J Chromatogr A. 2014 May 16;1342:54-62. doi: 10.1016/j.chroma.2014.03.050. Solvent systems with n-hexane and/or cyclohexane in countercurrent chromatography--
Physico-chemical parameters and their impact on the separation of alkyl hydroxybenzoates. Englert M1, Vetter W2.

26. Hydrophobic Compounds
Fig. 6. CCC-UV–vis chromatogram (450 nm) of the
separation of crude carrot extract with dual-mode
utilization (mobile phase and elution mode reversal).
Conditions:CCC-1000 with three serially connected
multilayer coils (1.6 mm i.d. PTFE tubing)and a total column
capacity of 325 mL and 10 mL sample loop; rotation
speed:1010 ± 10 rpm; solvent system: n-
H/benzotrifluoride/Ac (10:3.5:6.5,v/v/v); sample size: 100.2
mg; separation: dual mode CCC separation with tail-to-head
mode for 140 min at a flow rate of 2 mL/min and head-to-
tail elution for 60 min at a flow rate of 4 mL/min.
J Chromatogr A. 2015 Apr 3;1388:119-25. doi: 10.1016/j.chroma.2015.02.020.
Isolation of β-carotene, α-carotene and lutein from carrots by countercurrent chromatography with the solvent
system modifier benzotrifluoride. Englert M, Hammann S, Vetter W.