General introduction on the different instrument dependent methods that can be utilized in countercurrent chromatography

1. (Multiple) Instrument Dependent
Methods
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.)
J. Brent Friesen, Chemistry Professor, Dominican University jbfriesen@dom.edu

2. Multiple Instrument Dependent Methods
Scale up
See “Optimization Parameters”

3. Instrument Dependent Methods
Sequential CCC

4. Countercurrent Separations:
• 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

5. J Chromatogr A. 2007 Jun 1;1151(1-2):169-74. Epub 2007 Jan 11.
Advanced applications of counter-current chromatography in the isolation of anti-
tuberculosis constituents from Dracaena angustifolia.
Case RJ, Wang Y, Franzblau SG, Soejarto DD, Matainaho L, Piskaut P, Pauli GF.
Gradient-array counter-current fractionation of D. angustifolia and
corresponding anti-TB biochromatogram. Gradient-array CCC fractions from
separations using HEMWat 0 (1:1:1:1), HEMWat -3 (3:2:3:2) and HEMWat -5
(7:3:7:3). All fractionation steps utilized elution-extrusion CCC. Resulting
fractions are identified as F1–F11. Fractions marked with an X were combined
with the A-range from the HEMWat 0 run of the second subsample and
reinjected into HEMWat -5.
Sequential
CCS runs

6. DCM extract
HEMWat 8:2:8:2
HEMWat 5:5:5:5Inactive
HEMWat 5:5:5:5
HEMWat 7:3:5:5
HEMWat 7:3:6:4
HEMWat 8:2:8:2
Inactive
Compounds 1-7
Compounds 8-15
Compounds 16-23
Compounds 24 -36
Lower phase
Lower phase
Upper phase
Upper
phase
low K
low K
low K
high K
high K
high K
J Nat Prod. 2010 Apr 23;73(4):563-7. doi: 10.1021/np900674d.
Sesquiterpenes from Oplopanax horridus.
Inui T1, Wang Y, Nikolic D, Smith DC, Franzblau SG, Pauli GF.
Sequential
CCS runs

7. Fig. 2. Scheme of the five countercurrent chromatography (CCC)
fractionations which were conducted for the isolation of
20:45,11,14,17 (juniperonic acid) (CCC-1,2,3) and 20:35,11,14
(sciadonic acid) (CCC-1,4,5). Purities (P) and recoveries (R,relative to
initial amount) of the target fatty acids as well as the fractions and
total mass used for subsequent CCC fractionation are given next to the
arrows.
CCC-1 HMWat 700:350:4
CCC-2 HAc
CCC-3 HMWat 700:350:4
CCC-4 HAc
CCC-5 HepMWat 400:364:36
J Chromatogr A. 2015 May 15;1394:89-94. doi: 10.1016/j.chroma.2015.03.042. Isolation of two Δ5 polymethylene interrupted
fatty acids from Podocarpus falcatus by countercurrent chromatography.
Hammann S, Schröder M, Schmidt C, Vetter W.
Sequential
CCS runs

8. HMWat 340:240:1 (1% AgNO3)
J Chromatogr A. 2012 May 11;1237:96-105. doi: 10.1016/j.chroma.2012.03.033.
Investigation of unsaponifiable matter of plant oils and isolation of eight phytosterols by means of high-
speed counter-current chromatography. Schröder M, Vetter W.
Sequential
CCS runs

9. J Chromatogr A. 2015 May 15;1394:89-94. doi: 10.1016/j.chroma.2015.03.042. Isolation of two Δ5 polymethylene interrupted
fatty acids from Podocarpus falcatus by countercurrent chromatography.
Hammann S, Schröder M, Schmidt C, Vetter W.
Sequential CCS runs

10. HterMWat 5:5:2:3
CyterMWat 5:5:2:3
Figure 3. (Top) CCC separation in the first dimension (1D) with the four unresolved compounds a, b, c, and d employing solvent system I. The
three heart-cutting time windows are shaded in blue. (Bottom) 2D CCC/UV (254 nm) chromatogram with the corresponding three heart-cuts
shaded in green which resolved compounds a, b, c, and d from each other. The elution of the remaining compounds e−h is done in 1D from
156 to 204 min.
Anal Chem. 2015 Oct 20;87(20):10172-7. doi: 10.1021/acs.analchem.5b02859. Heart-Cut Two-Dimensional Countercurrent Chromatography with a Single Instrument. Englert
M, Brown L, Vetter W.

11. J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Nov 1; 1004:10-16. doi: 10.1016/j.jchromb.2015.09.017. One-step separation of nine structural analogues from Poria cocos (Schw.)
Wolf. via tandem high-speed counter-current chromatography. Zeng H, Liu Q, Yu J, Jiang X, Wu Z, Wang M, Chen M, Chen X.
Fig. 2. Chromatograms of T-
HSCCC separation. HEMWat
(5:5:6:4); stationary phase:
upper phase;: 850 rpm; 180 mg:
1.2 mL/min; :25◦C; Sf: 55.7%.
Sequential CCS runs

12. 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.
Sequential
CCS runs

13. 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.
Sequential CCS runs

14. Peak B
70 mg
Peak C
50 mgPetEMWat 1:1:1:1
{0.01 Cu(NO3)2}
180 mg
Peak A
60 mg
EWat
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Sep 15;1001:58-65. doi:
10.1016/j.jchromb.2015.07.046. Target-guided separation of antioxidants from
Semen cassia via off-line two-dimensional high-speed counter-current
chromatography combined with complexation and extrusion elution mode.
Zeng H1, Liu Q2, Wang M1, Jiang S1, Zhang L1, He X1, Wang J1, Chen X3.
Sequential CCS runs

15. Fig. 2. HSCCC chromatogramsof the n-butanol extract of aerial part of A. ilicifolius, along with the HPLC chromatograms of the crude n-butanol
extract and the fractions containing HBOA-Glc (1) and DIBOA-Glc (2) from HSCCC. (B) CCC 1, solvent system: ethyl acetate–n-butanol–0.5%NH4OH
(2:3:5, v/v/v); sample, 100mg n-butanol extract dissolved in 8mL of the mixture of ethyl acetate–n-butanol–0.5% NH4OH (2:3:5, v/v/v); (C) CCC 2,
solvent system, ethyl acetate–n-butanol–water (2:3:5, v/v/v); sample, 100mg extract dissolved in 8mL of the mixture of ethyl acetate–n-butanol–
water (2:3:5, v/v/v); (D) CCC 3, solvent system: ethyl acetate–n-butanol–0.5%NH4OH (2:3:5, v/v/v); sample: peak fraction of compounds 1 and 2 in
(C) was evaporated to dryness in vacuo, and dissolved in 8mL of the mixture ethyl acetate–n-butanol–0.5%NH4OH (2:3:5, v/v/v)., 260 mL, 850 rpm,
2.0 mL/min; Sf in (B), (C), and (D) were about 40%, 42%, and 36%, respectively.
J Chromatogr A. 2008 Sep 26;1205(1-2):177-81. doi: 10.1016/j.chroma.2008.08.010. Preparative isolation and purification of two
benzoxazinoid glucosides from Acanthus ilicifolius L. by high-speed counter-current chromatography. Yin H, Zhang S, Luo X, Liu Y.
SequentialCCSruns

16. Instrument Dependent Methods
Column Length

17. Fig. 2. The representative 1D CCC separations of (a) one, (b) two, (c) three columns
connented in series (namely channel A, A–B, A–B–C). 1, dihydrotanshinone I; 2, cryptotanshinone; 3,
tanshinone I; 4, 1,2-dihydrotanshinquinone; 5, tanshinone IIA; 6,
trijuganone B, 7, methyl tanshinonate, 8, danshenxinkun A. The solvent system of
hexane–ethyl acetate–methanol–water was used and prepared on demanded mode:
upper phase, hexane:ethyl acetate:methanol:water is 68.70:24.91:3.64:2.75 (v/v,
%); lower phase, hexane:ethyl acetate:methanol:water is 2.53:24.77:49.04:23.66
(v/v, %). The sample size was 300 mg and the rotation speed was kept at 500 rpm
and flow rate was 3 mL/min. The retention of stationary phase was controlled at 60%
by simutaneously pumped the upper phase and lower phase to fill the CCC column,
thus the injection mode was injection after equilibrium.
Journal of Chromatography A, 1323 (2014) 73– 81 Comprehensive multi-channel multi-dimensional
counter-current
chromatography for separation of tanshinones from Salvia miltiorrhiza
Bunge Jie Meng, Zhi Yang, Junling Liang, Hui Zhou, Shihua Wu,∗
Longer column à Better Resolution

18. Fig. 4. Separation of caffeine and vanillin with the Arizona L heptane–ethyl acetate–methanol–water 2:3:2:3 (v/v) liquid system. Reversed-phase mode: organic upper
stationary phase and aqueous lower mobile phase flown in the head to tail direction. Top chromatograms: column HPCCC Mini 19.6mL with a 0.8mm I.D. tubing coil,
2100 rpm, 2 mL/min. Sf = 38%, Rs = 2.0. Bottom chromatograms: column HPCCC Mini 20.8mL with a 1.6mm I.D. tubing coil, 1800 rpm, 5mL/min, Sf = 66%, Rs = 2.0. Injectionvolume 0.5
mL, concentration 1mg/mL in lower mobile phase. Detection UV at 254 nm. Left chromatograms shown in volume units. Right chromatograms shown in time units.
Berthod2009_JCA_1216_4169_SmallVolume
HPCCC
Mini 19.6mL
0.8 mm tubing
HPCCC
Mini 20.8mL
1.6 mm tubing
Longer column à Better Resolution

19. Fig. 5. Separation of nine GUESS solutes with HepEMWat 1:1:1:1 Organic upper stationary phase. Top chromatograms obtained
in 22min: column HPCCC Mini 19.6mL with a 0.8mmI.D. tubing coil, 2100 rpm, 2 mL/min. Sf = 46%. Bottom chromatograms
obtained in 13min: column HPCCC Mini 20.8mL with a 1.6mm I.D. tubing coil,1800 rpm, 5 mL/min, Sf = 72%. Injection volume
0.5 mL, concentration ∼1mg/mL (each) in lower mobile phase. Detection UV at 254 nm. The inset chromatograms are
enlargements of the early eluting peaks.. Carvone (O) retention time is indicated.
Berthod2009_JCA_1216_4169_SmallVolume
HPCCC Mini 19.6mL with
0.8mmI.D. tubing coil
HPCCC Mini 20.8mL
1.6mm I.D. tubing
Longer column à Better Resolution

20. Instrument Dependent Methods
Sample Prep

21. 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