Parameters to consider for the formulation of bi(tri)phasic solvent systems to be utilized in liquid/liquid 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. How does a separatory funnel work?
Liquid- Liquid Extraction
2. Add compound mixture (solutes)
3. Shake (vigorous mixing of phases)
4. Allow phases to separate (settling)
5. Physically separate layers
1. Formulate biphasic solvent mixture (system)
Solvent System = SS Phase System
6. Analyze layers for composition

3. Liquid- Liquid Extraction
Simple biphasic solvent systems are created by
mixing two immiscible liquids.
Organic/Aqueous
liquid/liquid separation

4. Biphasic solvent systems may have 2 or more solvents.
Liquid- Liquid Extraction
A common way of formulating solvent system is to start with 2
immiscible solvents and add one or more modifiers.
Chloroform/Water
Chloroform/Methanol/Water (ChMWat)
Hexane/Water
Hexane/Methanol/Water
Hexane/ Ethyl Acetate /Water
Hexane/ Ethyl Acetate /Methanol/Water HEMWat

5. Survey Results
23
133
223
10
3
2
3
4
5
6
number of solvents
number of solvents per SS
Friesen 2015 J Nat Prod v. 78 p. 1764
Liquid- Liquid Extraction

6. Ternary Phase Diagrams (for three-component solvent systems)
Liquid- Liquid Extraction

7. Solvent System Characterization
Berthod, A. (2002). Countercurrent Chromatography: The support-free liquid stationary phase. Wilson & Wilson's Comprehensive
Analytical Chemistry Vol. 38. Boston: Elsevier Science Ltd. pp. 1–397. ISBN 978-0-444-50737-2.

8. Fig. 3. (a) Chemical structure of benzotrifluoride and (b) ternary phase diagram of n-
hexane/benzotrifluoride/acetonitrile with two-phase region at 22◦C shown in grey
shading. Volumetric solvent composition for the CCC separation is represented as
ared triangle (50% n-hexane, 17.5% benzotrifluoride, 32.5% acetonitrile,
10:3.5:6.5).The tie-lines indicate that benzotrifluoride partitioned evenly between n-
hexane and acetonitrile. The composition of the upper and lower phases varied
extensively with the amount of benzotrifluoride in the system
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.
Solvent System Characterization

9. Liquid- Liquid Extraction
Biphasic solvent systems created by mixing two
or more immiscible liquids.
Alcohol/Ionic Aqueous or Acetonitrile/Ionic Aqueous
Aqueous Two Phase Solvent Systems (ATPS)
Polyethylene Glycol/Buffered Aqueous
Organic/Organic or “non-aqueous” (heptane/methanol)
Organic/Aqueous (ChMWat, HEMWat, etc…)

10. Unified Solvent Notation
Petroleum ether Pet DMSO So
Hexane H Acetonitrile Ac
Heptane Hep Isopropyl alcohol Iso
Cyclohexane Cy n-propanol Pro
Toluene Tol n-butanol Bu
Methyl tert-butylether ter Acetic acid Aa
THF Tet Ethanol Et
Diethyl ether De Methanol M
Ethyl acetate E Water Wat
Methyl acetate Me
Chloroform Ch
Dichloromethane Di
Acetone At
1. Solvent Abbreviations
Used Define Countercurrent Separation Solvent Systems

11. Unified Solvent System Notation
2. Order of polarity: least polar to most polar à HEMWat, ChMWat, terBuAcWat
4. Whole numbers à HEMWat 2:3:4:5
4a. No numbers needed for binary solvent systems
5b. Lowest common denominator? 5:5:5:5 or 1:1:1:1
6. pre-equilibration additives/modifiers in parentheses à terBuAcWat (0.7% TFA)
6a. What does concentration mean?
Concentration in a single component or overall mixture?
3. Volume ratios à HEMWat 2:3:4:5
6b. Modifier or solvent component? acetic acid, ionic liquids,
1. Solvent abbreviations
1a. Write out new or rare solvents
7. pH-zone refining and Ion-exchange (retainer - eluter)

12. HEMWat
http://2.bp.blogspot.com/-4jotcEHY_Iw/Tu7oPvHVX9I/AAAAAAAAAyk/fsircQR9n3A/s1600/workhorse_discing.jpg

13. The HEMWat SS Family
Biphasic solvent systems created by mixing two or more immiscible liquids.
organic/organic modifier/aqueous modifier/aqueous
+ low-cost
+ versatile and tunable (heptane or pet ether or limonene for hexane)
(ethanol for methanol)
+ good CCC/CPC performance (high Sf values)
Hexane/ Ethyl Acetate /Methanol/Water = HEMWat
- environmental impact / health concerns
- stability
- what about pH, shouldn’t we buffer this?
+ create solvent system families by systematic modification

14. The HEMWat SS Family
HEMWat solvent composition
0%
25%
50%
75%
100%
+8 +7 +6 +5 +4 +3 +2 +1 0 -1 -2 -3 -4 -5 -6 -7 -8
HEMWat number
Hexane
EtOAc
MeOH
Water
Friesen B & Pauli GF
G.U.E.S.S. to make Generally Useful Estimations of Solvent Systems in CCC.
Journal of Liquid Chromatography & Relat. Technologies 28: 2777-2806 (2005)
dx.doi.org/10.1080/10826070500225234

15. The HEMWat SS Family
Oka F, Oka H, Ito Y (1991) J Chromatogr A 538:99–105

16. Anal Bioanal Chem. 2005 Sep;383(2):327-340.
Alkane effect in the Arizona liquid systems used in countercurrent chromatography.
Berthod A1, Hassoun M, Ruiz-Angel MJ.
10.1007/s00216-005-0016-7
The HEMWat SS Family
Renault JH, Nuzillard JM, Intes O, Maciuk A (2002) In: Berthod A (ed) Countercurrent
chromatography, the support free liquid stationary phase (Comprehensive analytical chemistry,
vol. 38). Elsevier, Amsterdam, pp 49–83
The HEMWatSS family

17. The HEMWat SS Family
Dubant S, Mathews B, Higginson P, Crook R, Snowden M, Mitchell J. Practical
solvent system selection for counter-current separation of pharmaceutical
compounds. J Chromatogr A 2008; 1207: 190–192
The HEMWat SS family

18. A novel 9 × 9 map-based solvent selection strategy for targeted counter-current chromatography isolation of natural productsOriginal Research Article
Journal of Chromatography A, Volume 1400, 26 June 2015, Pages 27-39
Junling Liang, Jie Meng, Dingfang Wu, Mengzhe Guo, Shihua Wu doi:10.1016/j.chroma.2015.04.043
The HEMWat SS Family
8 x 8 = 64 possibilitiesThe HEMWat SS family

19. The HEMWat SS Family
J Chromatogr A. 2014 Nov 14;1368:116-24. doi: 10.1016/j.chroma.2014.09.064. Folding fan mode counter-current chromatography offers fast
blind screening for drug discovery. Case study: finding anti-enterovirus 71 agents from Anemarrhena asphodeloides.
Liu M, Tao L, Chau SL, Wu R, Zhang H, Yang Y, Yang D, Bian Z, Lu A, Han Q, Xu H8.

20. 461
261
232
172
104
42
35
33
24
23
22
18
8
5
3
2
2
1
water
ethyl acetate
methanol
hexane
n-butanol
acetonitrile
ethanol
chloroform
pet ether
heptane
methyl t-butyl ether
isopropanol
dichloromethane
acetone
ethyl ether
cyclohexane
carbon tetrachloride
THF
Survey Results
Friesen2015_JNP_78_1764_countercurrent
Solvents used in Biphasic SSs
97% had water
35% HEMWat
16% similar to HEMWat
The HEMWat SS Family

21. Solvent System Properties
http://us.hola.com/imagenes/viajes/2011071553613/chicago-moderno-estados-unidos/0-181-586/a_009GenSM-a.jpg

22. Solvent System Properties
Polarity
Selectivity
Friesen, J. B.; Ahmed, S.; Pauli, G. F. Qualitative and quantitative evaluation of solvent systems for countercurrent separation. Journal of
Chromatography A 2015, 1377, 55–63 DOI: 10.1016/j.chroma.2014.11.085.
Will my chromatography method position the target
compounds with a reasonable VR (tR)?
Not in void volume.
Not highly retained on the column
Can molecules with similar polarity be separated in
that region with a reasonable VR?
match between solvent system and solute (polarity scales such as LogPo/w ,
Reichardt’s Dye scale, and eluotropic sequences )

23. Polarity
1. (Linear combination of)
Eluotropic sequence parameters
2. Solvatochromic (Reichardt’s) Dye
3. GUESSmix method
4. Phase Metering Apparatus

24. (Linear combination of) Eluotropic sequence parameters
Journal of Chromatography A Volume 1393, 8 May 2015, Pages 47–56 Systematic and practical solvent system selection strategy based on the nonrandom two-liquid segment
activity coefficient model for real-life counter-current chromatography separation Da-Bing Ren, Lun-Zhao Yi, Yan-Hua Qin, Yong-Huan Yun, Bai-Chuan Deng, Hong-Mei Lu, Xiao-
Qing Chen, Yi-Zeng Liang,

25. Journal of Chromatography A Volume 1393, 8 May 2015, Pages 47–56 Systematic and practical solvent system selection strategy based on the nonrandom two-liquid segment
activity coefficient model for real-life counter-current chromatography separation Da-Bing Ren, Lun-Zhao Yi, Yan-Hua Qin, Yong-Huan Yun, Bai-Chuan Deng, Hong-Mei Lu, Xiao-
Qing Chen, Yi-Zeng Liang,
(Linear combination of) Eluotropic sequence parameters

26. Solvent X Y- Y+ Z
acetonitrile 0.018 0.131 0.883 0.000
chloroform, 0.393 0.0 0.167 0.000
ethyl acetate 0.3339 0.058 0.441 0.000
ethanol 0.251 0.030 0.000 0.630
n-butanol 0.425 0.004 0.0 0.490
methanol 0.090 0.139 0.0 0.594
methyl tert-butyl ether 0.483 0.105 0.142 0.000
hexane 1.000 0.000 0.000 0.000
heptane 1.152 0.000 0.000 0.000
water 0.000 0.000 0.000 1.000
Table S1. The NRTL-SAC parameters of the used solvents in this work.
Journal of Chromatography A Volume 1393, 8 May 2015, Pages 47–56 Systematic and practical solvent system selection strategy based on the nonrandom two-liquid segment
activity coefficient model for real-life counter-current chromatography separation Da-Bing Ren, Lun-Zhao Yi, Yan-Hua Qin, Yong-Huan Yun, Bai-Chuan Deng, Hong-Mei Lu, Xiao-
Qing Chen, Yi-Zeng Liang,
Solute X Y- Y+ Z
Nicotinic acid 0.093 0.000 0.272 0.588
3,4-dihydroxybenzoic acid 0.338 0.728 0.708 0.742
Chlorogenic acid 0.142 1.074 1.086 1.416
Antipyrine 0.112 0.220 0.446 0.594
(Linear combination of) Eluotropic sequence parameters
Polarity

27. Polarity
2. Solvatochromic (Reichardt’s) Dye

28. Hexanes Toluene t-BME Limonene Dichloromethane
Dichloromethane Ethyl Acetate Acetone n-Butaniol Isopropanol
Isopropanol Acetonitrile Ethanol Methanol Water

29. Solvatochromic (Reichardt’s) Dye
https://upload.wikimedia.org/wikipedia/commons/thumb/2/28/Reichardt_dye_structure.svg/220px-Reichardt_dye_structure.svg.png

30. Solvatochromic (Reichardt’s) Dye
400
500
600
wavelengthinnm
Upper Phase
Lower Phase
Reichardt’s dye lambda max values in the upper and lower phases of HEMWat solvent systems. Some
values could not be determined due to insufficient solubility of the dye.
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085

31. 500
600
wavelength in nm
Ch(Di)Mwat Solvent System
DiMWat
ChMWat
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085
Fig. 5. Reichardt’s dye lambda
max values in the lower phases of
ChMWat and DiMWat solvent
systems.
Solvatochromic (Reichardt’s) Dye

32. Polarity
3. GUESSmix

33. 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

34. Solvent Systems Surveyed
hexane
ethyl
acetate
methanol
chloroform
dichloromethane
water
methyl
t-butyl
ether
ACN
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085

35. GUESSmix polarity index (GUPI)
0
10
20
HepM 10:10
HterAcWat 8:2:8:2
HBuMWat 5:5:5:5
HEMWat 7:3:6:4
HEMWat 8:2:8:2
HEMWat 5:5:5:5
terBuMWat 5:5:5:5
HEMWat 4:6:4:6
DiMWat* 10:7:3
DiMWat* 10:6:4
terAcWat 7:3:10
terAcWat 7:6:7
HterAcWat 3:7:3:7
HterAcWat 6:10:1:4
HEMWat 1:9:1:9
HEMWat 3:7:3:7
ChWat* 10:10
ChMWat* 10:6:4
ChMWat* 10:4:6
ChMWat* 10:2:8
DiWat* 10:10
DiMWat* 10:4:6
DiMWat* 10:2:8
terAcWat 5:5:5
terAcWat 5:5:10
EBuWat 8:2:10
HterAcWat 5:5:5:5
EBuWat 4:6:10
terAcWat 8:2:10
number of GUESSmix compounds
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085
Fig. 1. Graphic representation of the GUESSmix polarity index (GUPI) of the solvent systems analyzed in this study. GUPI is the
number of GUESSmix compounds with K values greater than 1 divided by the number of GUESSmix compounds with K values
less than or equal to 1 in a reversed-phase elution method (aqueous or polar phase mobile). *The ChMWat and DiMWat solvent
systems are represented as the inverse of the original K values.

36. Polarity
4. Phase Metering Apparatus

37. Phase Metering Apparatus
280000
280500
281000
281500
282000
282500
0 0.5 1 1.5 2
Log(Delectric Constant [literature values])
Pauli GF, Pro S, Chadwick L, Burdick T, Pro L, Friedl W, Novak, N, Maltby J, Qiu, F, Friesen JB Real-Time Volumetric Phase Monitoring Advances
Chemical Analysis by Countercurrent Separation Analytical Chemistry 87:7418-7425 (2015)
dx.doi.org/10.1021/acs.analchem.5b01613

38. Phase Metering Apparatus
300,500
302,500
304,500
2
PMA
values
time in min
301,500
302,500
303,500
304,500
2 6
PMA
values
time in min
HEMWat
5:5:5:5
UP
HEMWat
5:5:5:5
UP/LP
1:1
HEMWat
5:5:5:5
LP
HEMWat
5:5:5:5
LP
HEMWat
5:5:5:5
UP
Pauli GF, Pro S, Chadwick L, Burdick T, Pro L, Friedl W, Novak, N, Maltby J, Qiu, F, Friesen JB Real-Time Volumetric Phase Monitoring Advances
Chemical Analysis by Countercurrent Separation Analytical Chemistry 87:7418-7425 (2015)
dx.doi.org/10.1021/acs.analchem.5b01613

39. Phase Metering Apparatus
300000
302000
304000
306000
upper
lower
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085

40. Phase Metering Apparatus
300950
301950
302950
303950
304950
ChMWat DiMWat
Upper
Lower
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085

41. Phase Metering Apparatus
301900
302900
303900
HEMWat 10:0:10:0
HEMWat 9:1:9:1
HEMWat 8:2:8:2
HEMWat 7:3:7:3
HEMWat 7:3:6:4
HEMWat 6:4:6:4
HEMWat 7:3:5:5
HEMWat 6:4:5:5
HEMWat 5:5:5:5
HEMWat 4:6:5:5
HEMWat 3:7:5:5
HEMWat 4:6:4:6
HEMWat 3:7:4:6
HEMWat 3:7:3:7
HEMWat 2:8:2:8
HEMWat 1:9:1:9
HEMWat 0:10:0:10
ChMWat 10:0:10
ChMWat 10:1:9
ChMWat 10:2:8
ChMWat 10:3:7
ChMWat 10:4:6
ChMWat 10:5:5
ChMWat 10:6:4
ChMWat 10:7:3
DiMWat 10:0:10
DiMWat 10:1:9
DiMWat 10:2:8
DiMWat 10:3:7
DiMWat 10:4:6
DiMWat 10:5:5
DiMWat 10:6:4
DiMWat 10:7:3
EBuWat 10:0:10
EBuWat 8:2:10
EBuWat 6:4:10
EBuWat 4:6:10
EBuWat 2:8:10
EBuWat 0:10:10
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085
Fig. 3. Averages of the upper and lower phase permittivity
values measured by the phase metering apparatus for EBuWAt,
DiMWat, ChMWat, and HEMWat.

42. Selectivity
http://www.chocolati.com/new/images/P/24pcmix.jpg
Solvent System Properties

43. Selectivity
60
160
PW
PW+SS+PWSS
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085
Fig. 6. Selectivity parameters calculated for HEMWat solvent systems. Pairwise analysis calculated the ratio of K values for
every pairwise combination. A ratio of greater than 5/3 or less than 3/5 is considered to be adequate for the resolution of two
analytes. The number of resolved pairs is represented by αip. The number of GUESSmix analytes in the sweet spot is
represented by Nsw. The number of resolved pairs in the sweet spot is represented by αsw. The sum of these three
parameters (αip + Nsw + αsw), produced a selectivity score .

44. Selectivity
140
190
PW
PW+SS+PWSS
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085
Fig. 6. Selectivity parameters calculated for DiMWat solvent
systems. Pairwise analysis calculated the ratio of K values for
every pairwise combination. A ratio of greater than 5/3 or less
than 3/5 is considered to be adequate for the resolution of two
analytes. The number of resolved pairs is represented by αip.
The number of GUESSmix analytes in the sweet spot is
represented by Nsw. The number of resolved pairs in the
sweet spot is represented by αsw. The sum of these three
parameters (αip + Nsw + αsw), produced a selectivity score .

45. Selectivity
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085
Fig. 6. Selectivity parameters calculated for terAcWat solvent
systems. Pairwise analysis calculated the ratio of K values for
every pairwise combination. A ratio of greater than 5/3 or less
than 3/5 is considered to be adequate for the resolution of two
analytes. The number of resolved pairs is represented by αip.
The number of GUESSmix analytes in the sweet spot is
represented by Nsw. The number of resolved pairs in the
sweet spot is represented by αsw. The sum of these three
parameters (αip + Nsw + αsw), produced a selectivity score .

46. Polarity and Selectivity
2D Reciprocal (Shifted) Symmetry Plots
Mismatched Polarity
Similar Polarity and
Selectivity
2.7 4 8
8
2.7 4 8
8
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085

47. Polarity and Selectivity
2D Reciprocal (Shifted) Symmetry Plots
Similar Polarity with
Different Selectivity
Development and
identification of orthogonal
solvent systems is the key to
the use of successive
Countercurrent Separation
steps in purification.
2.7 4 8
8
Friesen JB, Ahmed S, Pauli GF
Qualitative and Quantitative Evaluation of Solvent Systems for Countercurrent Separation
Journal of Chromatography A 1377: 55-63 (2015)
dx.doi.org/10.1016/j.chroma.2014.11.085

48. 1
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.5 1 1.5 2 2.5 3 3.5 4
HBuMWat 5:5:5:5
HterAcWat5:5:5:5
2.7
4
8
8
2.7 4 8
8
ChMWat 10:7:3
DiMWat10:7:3
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.5 1 1.5 2 2.5 3 3.5 42.7 4 8
8
2.7
4
8
8
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.5 1 1.5 2 2.5 3 3.5 4
DiMWat10:6:4
HEMWat 4:6:4:6
2.7 4 8
8
2.7
4
8
8
R
C
V/A
M
E
Z
UN
F
Q
D
DiMwat10:6:4
HterAcwat 4:6:4:6
0
0.5
1
1.5
2
2.5
3
3.5
4
0 0.5 1 1.5 2 2.5 3 3.5 42.7 4 8
8
2.7
4
8
8
R
C
M
E
Z
U
Q
AV
N
F
D
A B
C D

49. GUESSmix Polarity Matching

50. KD
interval
s
0
≤ KD <
0.0625
0.062
5
≤ 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 <
∞
-8
rXHTDC
GRFQUA
VNMEZ
OI Y b
-7
rXHTDC
GRFQUA
VNE
MZ O I Y b
-6
rXHTDC
GRFQUA
VNE
MZ O I Yb
-5
rXHTD
CGRAV
FQU
NE
MZ O I Yb
-4
rXHTD
CGR
AV
FQ
U
NE M Z O I Yb
-3
rXHTD
CGR
FQ
U
AVN E MZ O I Yb
-2
rXHTD
CGR
FQ
U
AVN ME Z O I Yb
-1
rXHTD
CG
RFQU AVN MEZ O I Yb
0 rXHTG DR CF
QUA
V
N ME Z O I Yb

51. 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 <
∞
0
rXH
TG
DR CF
QUA
V
N ME Z O I Yb
+1
rXH
T
GR
DC
F
QUA
V
NM EZ O I Yb
+2 r XHT DCG RF
QUA
V
NM
EZ
O I Yb
+3 r XHT DCG RF
QUA
V
M
NE
Z
O IYb
+4
rXH
T
DCG RFU
QA
V
NM
EZ
O IYb
+5 r XHT DC G
RF
UA
V
M QZ
NEOIY
b
+6 r XHT D CG F
RU
AV
M Z
QNEO
IYb
+7 r XHT D CG F
RUA
V
MZ
QNEO
IYb
+8
rXH
T
D CG
FRUA
VM
Z
QNEO
IYb
Friesen JB, Pauli GF. Performance characteristics of countercurrent separation in analysis of natural products of agricultural significance. J. Agric. Food Chem., 56, 19-28, 2008

52. EBuWat Solvent System Family
Ethyl Acetate / n-Butanol / Water
0
20
40
60
80
100
+5 +4 +3 +2 +1 0 -1 -2 -3 -4 -5
solvent system number
%
Ethyl Acetate
Butanol
Water

53. Solvent System Maps
Friesen, J.B. Pauli, G.F. Journal of Chromatography A 1151: 51-59 (2007)
Sweet Spot
FVAUMNE
RZQOIYb
GCDrTXH10:0:10
FVAUMNE
RZQOIYb
GDCXHTr8:2:10
FVAUMNE
RZQOIYb
GDCTXHr6:4:10
FVAUMNE
RZQOIYb
GCDTXHr4:6:10
FVAUMNE
RZQOIYb
GCDTXHr2:8:10
VAUMNER
ZQOIYb
FGCDTXHr0:10:10
32
≤ KD <
∞
16
≤ KD <
32
8
≤ KD <
16
4
≤ KD <
8
2
≤ KD <
4
1
≤ KD <
2
0.5
≤ KD <
1
0.25
≤ KD <
0.5
0.125
≤ KD <
0.25
0.062
5
≤ KD <
0.125
0
≤ KD <
0.0625
KD
intervals
Sweet Spot
EBuWat

54. KD
intervals
0
≤ KD <
0.0625
0.063
≤ 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 <
∞
HEMW
at 0
rXHTG DR CF QUAV N ME Z O I Yb
DEMW
at 0
rXHT G D C
FUV
A
RQ ZMNE OI Yb
DEMWat 0 (5:5:5:5)
HEMWat 0 (5:5:5:5)
FDR
A
O Yb
Z
E
M
NA
VU
X
H
T
G
r
C
Q
I
0 0.25 0.5 0.75 1 1.33 2 4 ∞K'(1)
∞
A
280nm
230nm
X
H
T
r
G
D
C
F
U
V
A
R
Q
ZMNE
OI
Yb
0 0.25 0.5 0.75 1 1.33 2 4K'(1)
SSMap
ReSPlotReSPlot
Pauli, G.; Pro, S.; Friesen, J. B.
Countercurrent Separation of Natural Products. J. Nat. Prod. 2008, 71, 1489-1508

55. terAcWat
system #
relative proportions of solvents phase
ratiot-butyl
methyl
ether
acetonitrile water
-5 10 0 10 45/55
-4 9 1 10 43/57
-3 8 2 10 41/59
-2 7 3 10 38/62
-1 6 4 10 36/64
0 5 5 10 34/66
+1 4 6 10 33/67
Solvent System Maps

56. QNEOI
Yb
MZAUFVRGCDXHTr
4:6:10
ZQNE
OIYb
UFMVARGCDrTXH
5:5:10
ZQNE
OIYb
UFMVARCDGrTXH
6:4:10
ZQNE
OIYb
UFMVARGCDrTXH
7:3:10
ZQNE
OIYb
UFMVARDGCrTXH
8:2:10
ZQNE
OIYb
UFMRVACDGrTXH
10:0:10
32
K <
16
K <
32
8
? KD <
16
4
K <
8
2
? KD <
4
1
? KD <
2
0.5
? KD <
1
0.25
? KD <
0.5
0.125
K <
0.25
0.0625
K <
0.125
0
KD <
0.0625
K
bins
QNEOI
Yb
MZAUFVRGCDXHTr
4:6:10
ZQNE
OIYb
UFMVARGCDrTXH
5:5:10
ZQNE
OIYb
UFMVARCDGrTXH
6:4:10
ZQNE
OIYb
UFMVARGCDrTXH
7:3:10
ZQNE
OIYb
UFMVARDG
ZQNE
OIYb
UFMRVA
32
Ki <
¥
16
Ki <
32
8
Ki <
16
4
Ki <
8
2
Ki <
4
1
Ki <
2
0.5
Ki <
1
0.25
Ki <
0.5
0.125
Ki <
0.25
0.0625
£ Ki
0.125
0
£ Ki
0.0625
i
Sweet
Spot
terAcWat
SolventSystemMap
of GUESSmixstandards
£ £ £ £ £ £ £ £ £
G
A
V
C
C
R
X
DN
COOHOH
HOOC
OH
OH
O
O
OH
OH
N
N N
N
O
O
CH3
CH3
H3C
OHO
HO
OH
HO
O
OH
N
H
N
O
H3CO OCH3
O
H3CO
OCH3
OCH3
OCH3
OH
OH
O
O
O
CH3
O OHO
OCH3
OH
OH
N
COOHOH
HOOC
OH
OH
O
O
OH
OH
N
N N
N
O
O
CH3
CH3
H3C
OHO
HO
OH
HO
O
OH
N
H
N
O
H3CO OCH3
O
H3CO
OCH3
OCH3
OCH3
OH
OH
O
O
O
CH3
O OHO
OCH3
OH
OH

57. HterAcWat
system #
relative proportions of solvents phase
ratiohexane tBME acetonitrile water
-8 10 0 10 0 40/60
-7 9 1 9 1 41/59
-6 8 2 8 2 41/59
-5 7 3 7 3 43/43/14
-4 7 3 6 4 45/24/31
-3 6 4 6 4 44/28/28
-2 7 3 5 5 47/10/43
-1 6 4 5 5 46/14/40
0 5 5 5 5 51/11/38
+1 4 6 5 5 63/37
+2 3 7 5 5 66/34
+3 4 6 4 6 58/42
+4 3 7 4 6 59/41
+5 3 7 3 7 55/45
+6 2 8 2 8 50/50
+7 1 9 1 9 49/51
+8 0 10 0 10 45/55

58. 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 <
∞
10:0:10:0
rTXHGDC
RFVUAQN
EMZ
O I Y b
8:2:8:2
rTXHGDC
RFVUAQN
E MZ O I Yb
4:6:5:5 rTXH DCG RF AVU Q M ZN
E
OIYb
4:6:4:6 rTXH DCG RF AVU Q M ZN
E
OIYb
3:7:3:7 rTXH DC G RF
AV
U
M ZQ
NE
OIYb
2:8:2:8 rTXH DC G RA FVU M
ZQNE
OIYb
0:10:0:10 rTXH DCG RAV FUM
ZQNE
OIYb
Sweet Spot
HterAcWatSolvent System Map

59. Comparing SS in different families
r T X H C D G R V A F U M Z Q N E O I Y b
c) terAcWat6:4:10
polar r T X H D C G V A F U M N E R Z Q O I Y b nonpolar
a) EBuWat
r T X H C D G R F V A U Q M Z N E O I Y b
d) HterAcWat4:6:4:6
r T X H C D G R V A F U M Z Q N E O I Y b
b) terAcWat
Friesen JB, Pauli GF. Rational development of solvent system families in counter-current
chromatography. J. Chromatogr. A, 1151, 51-59, 2007;