Different solvent delivery methods in Counterurrent Chromatography

Solvent Delivery Methods
A. On Demand Solvent Mixing
B. Gradients
C. pH-Zone Refining
D. Ion Exchange

A. On Demand Solvent Mixing
Anal Bioanal Chem (2005) 383: 327–340 DOI 10.1007/s00216-005-0016-7
A. Berthod . M. Hassoun . M. J. Ruiz-Angel
Alkane effect in the Arizona liquid systems used in countercurrent chromatography

B. Gradients
1. Step gradient
1a. Polarity-Adjusted SSs
1b. Differently Formulated SSs
2. Linear gradient
3. Three phase gradient

Figure 3. HSCCC chromatogram of crude
extract of ChanSu. HEMWat (4:6:2:4, 4:6:2.5:4
and 4:6:3.2:4 v/v) in stepwise elution;
stationary phase: upper organic phase of
4:6:2:4 by volume; mobile phase: lower
aqueous phase of 4:6:2:4 in 0–200min, lower
aqueous phase of 4:6:2.5:4 by volume in 200–
400 min, and lower aqueous phase of
4:6:3.2:4 by volume in 400–700min, flow rate
1.5mL/min; revolution speed, 800 rpm;
detection wavelength, 296 nm; separation
temperature, 201C; sample size, 80 mg crude
sample dissolved in 5mL of the upper phase
and 5mL of the lower phase. Retention of the
stationary phase: 56%.
Li2010_JSS_33_1325_bufadienolides
B.1a. Step Gradient: Polarity Adjusted

Mar. Drugs 2015, 13, 354-365; doi:10.3390/md13010354 Preparative Separation of Sulfur-Containing Diketopiperazines from Marine Fungus Cladosporium sp. Using High-Speed Counter-Current
Chromatography in Stepwise Elution Mode Binbin Gu, Yanying Zhang, Lijian Ding, Shan He, Bin Wu, Junde Dong, Peng Zhu, Juanjuan Chen, Jinrong Zhang and Xiaojun Yan
Figure 3. marine fungus Cladosporium sp. using stepwise elution with solvent systems A and B. Solvent system A: HEMWat
(1:1:1:1), solvent system B: HEMWat (2:1:2:1); stationary phase: upper organic phase of solvent system A; mobile phase: 460 mL
lower aqueous phase of solvent system A and 240 mL lower aqueous phase of solvent system B; column capacity, 300 mL, 900
rpm, 25 °C;, 2.0 mL/min; detection, 254 nm; sample size, 300 mg Sf, 68%. Peaks: 1 = cladosporin A (1) + haematocin (2); 2 =
cladosporin B (3).

Sf = 0.75
Aqueous stationary
5 mL/min
1200 rpm
J Sep Sci. 2013 Apr;36(8):1362-1369. doi: 10.1002/jssc.201201033.
Gradient elution method in centrifugal partition chromatography for
the separation of a complex sophorolipid mixture obtained from
Candida bombicola yeasts. Kotland A, Hadef I, Renault JH, Hamzaoui
M, Martinez A, Borie N, Guilleret A, Reynaud R, Hubert J.

B.1b. Step Gradient: Differently Formulated
The stationary phase was water saturated with n-
butanol and ethyl acetate. The stepwise elution was
performed with the following solvents:
n-hexane–ethyl acetate (1:1) for 80 min,
n-hexane– ethyl acetate (1:2) for 80 min,
n-hexane–ethyl acetate (1:4) for 80 min,
ethyl acetate for 80 min,
n-butanol–ethyl acetate (1:4) for 80 min,
n-butanol–ethyl acetate (1:2) for 80 min,
n-butanol– ethyl acetate (2:2) for 80 min,
and n-butanol–ethyl acetate (2:1) for 80 min.
The effluent was collected in 20-ml fractions by a
fraction collector.
All fractions were assayed for antioxidant activity. The
fractions were combined into seven larger fractions
(components) on the basis of their antioxidant activity
Fig. 1. Antioxidant activity-chromatogram
of fractions obtained from HSCCC
separation of 600 mg ethanolic M.
sempervirens leaf extract.
Du2012_FC_131_1181_Mucuna

B.2. Linear Gradient
Figure 2. The representative CCC profiles for separation of podophyllotoxin from cytotoxic fraction II with (A) and (B) isocratic elution, and (C) linear and
(D) step-gradient elution by using the system of HEMWat. 1, 4-demethylpodophyllotoxin, 2, -peltatin; 3, podophyllotoxin, 4, β-peltatin. CCC separation
conditions. (A) Isocratic elution with 4:6:3:7, upper phase as stationary phase and lower phase as mobile phase, retention of the stationary phase,
61.11%. (B) Isocratic elution with 4:6:4:6, upper phase as stationary phase and lower phase as mobile phase, retention of the stationary phase, 59.26%.
(C) Lineargradient elution, upper phase of 4:6:3:7 as stationary phase, mobile phase: 0–120 min, 100% of the lower phase of 4:6:3:7; 120–255 min, the
lower phase of 4:6:3:7 from 100% to 0 and the lower phase of 4:6:4:6 from 0 to 100%; after 255 min, 100% of the lower phase of 4:6:4:6. (D) Step-
gradient elution, upper phase of 4:6:3:7 as stationary phase, mobile phase: 0– 120 min, the lower phase of 4:6:3:7; after 120 min, the lower phase of
4:6:4:6. Flow rate, 2 mL/min. Rotation speed, 900 rpm.
Detection, 254 nm. Sample solution, 77 mg of the fraction II was dissolved in a solution composed of the upper and lower phases (1:1, v/v, 4 mL of the
total volume). Yields and purity, (C) 1, 2.7 mg with 86.35%, 2, 8.8 mg with 99.94%, 3, 21.1 mg with 96.9%, 4, 27 mg with 99.02%; (D) 1, 2 mg with
89.19%, 2, 8.1 mg with 99.92%, 3, 20.2 mg with 96.12%, 4, 29.1mg with 98.78%.
Yang2013_JSS_36_1022_podophyllotoxins

3-phase gradient
Filled with LP. 1000 rpm. The sample solution was loaded into the column by pumping UP0 from 0 to 20 mL/min in 3 min in the ascending mode. The
UP0 was pumped for 50 min after the release of the dead volume to ensure the elution of all hydrophobic compounds. The moderately polar MP was
then pumped for 33 min to elute compounds with a medium hydrophobicity. Finally, in order to recover the most hydrophilic compounds retained at
the head of the column, the role of the two liquid phases was switched by pumping the aqueous LP as the mobile phase in the descending mode at 20
mL/min.
Phytochem Anal. 2013 Jul-Aug;24(4):367-73. doi: 10.1002/pca.2418. Stepwise elution of a three-phase solvent system in centrifugal partition extraction: a
new strategy for the fractionation and phytochemical screening of a crude bark extract. Hamzaoui M, Renault JH, Nuzillard JM, Reynaud R, Hubert J.

pH zone refining
Journal of Chromatography A, 1065 (2005) 145–168 Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography Yoichiro Ito
“The greatest advantage of the method is its large sample loading capacity,
which exceeds 10-fold that of the standard HSCCC in the same separation
column. In addition, the method provides various special features such as
yielding highly concentrated fractions, concentrating minor impurities for
detection, and allowing the separation to be monitored by the pH of the
effluent when there are no chromophores.
Selection of solvent systems and preparation of the sample
are quite different from those used in the standard HSCCC
technique.”

pH zone refining
Fig. 4. Mechanism of sharp peak formation. The acid analyte is always
confined around the sharp retainer border and elutes as a sharp peak
with the retainer acid.
R
C
O
O
H N
H
H
H
R
C
O
O
R
C
O
OH
R
C
O
OH
N
H
H
H
N
H
H
H
H N
H
H
H
F3C
C
O
OH
H N
H
H
H
R
C
O
OH
F3C
C
O
O
H N
H
H
HH N
H
H
H
H N
H
H
R
C
O
OH
O HO H O H
O H
H

pH zone refining
Fig. 5. Model experiment to demonstrate the
mechanism of pH-zone refining CCC.(B) pH-zone
formation of three analytes in the column (upper
diagram), and the elution profile of the analytes
(lower diagram).
pKa (TFA) < pKa (S1) < pKa (S2) < pKa (S3)

pH zone refining
Rule 13: For an acidic analyte follow these steps:
(1) A 2 ml volume of each phase and 5 microL of NH4OH (ca. 28% NH3
stock solution) (eluter) is delivered into a test tube (13mm×100 mm)
or bring the pH above 10.
(2) Add a small amount of the analyte (so that no significant change is
made in pH), apply a stopper and vortex several times to equilibrate
the contents.
(3) Measure the analyte concentration in the upper and the lower
phases to obtain KU/L value or Kbase.
(4) If Kbase<< 1, add TFA (retainer) (ca. 20 mM) to the contents to bring
the pH to around 2, and reequilibrate the contents by vortexing.
(5) Using the procedure in Step (3), obtain Kacid, and if Kacid >>1, the
solvent composition is suitable for separation.
(6) If Kbase is not small enough, repeat the whole procedure using a less
polar solvent system such as HEMWat, 1:1:1:1 in Table 1 and move
upward.
(7) If Kacid is not large enough, repeat the whole procedure using a
more polar solvent system of terAcWat (2:2:3) in Table 2 and
downward.
Rule 14: For a basic analyte, substitute HCl for NH4OH at Step (1) to
test Kacid<<1, and substitute triethylamine for TFA at Step (4) to test
Kbase >> 1.
Rule 15: Start with equal molar
concentrations of retainer and eluter
such as 10–20mM each.
Rule 16: If the settling time of the sample
solution is lengthy, further dilution of the
sample is recommended.
Rule 17: Routinely measure the pH of
the sample solution before applying it to
the column.
Rule 18: Leave a small amount of the
stationary phase free of hydrophobic
counterions at the end of the column.

1. Separation of Acids
C. pH zone refining

C.1. pH zone refining
Separation of D&C Orange No. 5 by pH-zone-refining CCC. Experimental conditions: Apparatus: type-J coil
planet centrifuge with a multilayer coiled column (1.6 mm ID and 320 ml capacity); solvent system:
diethyl ether–acetonitrile–0.01 M aqueous ammonium acetate (pH 9 adjusted with ammonia) (4:1:5,
v/v), mobile phase: lower aqueous phase; sample: 5 g of D&C Orange No. 5 dissolved in 80 ml of solvent
(40 ml each phase); retainer: TFA 200 microL in the sample solution; flow rate: 3 ml/min; detection:206
nm; revolution: 800 rpm.
Journal of Chromatography A, 1271 (2013) 71– 85 Journal of Chromatography A pH-zone-refining counter-
current chromatography: Origin, mechanism, procedure and applications
Yoichiro Ito
https://cdn.shopify.com/s/files/1/0419/3361/products/265_featured_large.jpeg?v=1432937322

Fig. 3. PZRCCC chromatograms for the separation of the crude extract from the U. longissima Ach. Experimental conditions: (a)
solvent system: PEMH (5:5:2:8, v/v); 10 mM TFA in upper organic phase and 10 mM NaOH in lower aqueous phase; (b) solvent
system: PEMH (5:5:3:7, v/v); 10 mM TFA in upper organic phase and 10 mM NaOH in
lower aqueous phase; (c) solvent system: PEMH (5:5:3:7, v/v); 10 mM TFA in upper organic phase and 10–20 mM NaOH in lower
aqueous phase; revolution speed: 850 rpm; flow-rate: 2 mL/min; sample size: 1.2 g; UV detection wavelength: 254 nm.
J Chromatogr A. 2016 Jan 4;1427:96-101. doi: 10.1016/j.chroma.2015.12.016. Optimisation and establishment of separation conditions of organic acids
from Usnea longissima Ach. by pH-zone-refining counter-current chromatography: Discussion of the eluotropic sequence. Sun C, Liu F, Sun J, Li J, Wang X.

CCC chromatogram for the crude extract from G.
lucidum. Two-phase solvent system: PetEMWat
(3:5:3:5 and 4:5:4:5), flow rate:
5.0 ml/min, revolution speed: 500 rpm, detection
wavelength: 254 nm, sample size: 2 g crude extract
dissolved in 15 ml upper phase (3:5:3:5).
Fig. 4. Separation of mixture from peaks 3 and 4 (Fig. 3) by pH-zone-refining CCC. Above: separation of peak 3; the latter: separation of peak 4. Experimental conditions:
apparatus, three multiplayer coils separation column connected in series (I.D. of the tubing = 1.6 mm, total volume = 260 ml); solvent system: chloroform–methanol–water
(13:7:4), 22 mM NH4OH in upper aqueous stationary phase and 11 mM CF3COOH in lower organic phase; sample, 568 mg mixture from peak 3 were dissolved in 10 ml
stationary with NH4OH added; flow rate: 2.0 ml/min; revolution speed: 800 rpm; detection wavelength: 254 nm.
Food Chemistry Volume 130, Issue 4, 15 February 2012, Pages 1010–1016
Preparative isolation of triterpenoids from Ganoderma lucidum by counter-current chromatography combined with pH-zone-refining
Chun-Ru Cheng, Yi-Feng Li, Ping-Ping Xu, Rui-Hong Feng, Min Yang, Shu-Hong Guan, , De-An Guo

Separation of 500mg fatty acids from sunflower oil
with pHzone- refining CCC in a normal displacement
(20 mM NH3
(retainer) and 35 mM TFA (eluter)) and
b reverse displacement mode (20 mM TFA
(retainer) and 35 mM NH3 (eluter)) with UV/Vis signal
and pH curve obtained by measuring each fraction.
Experimental conditions: CCC-1000 instrument 60 mL
coil volume and 2 mL/min; 1,000 rpm;
solvent system: HAcMWat (40/70/14/
5, v/v) with TFA in the upper
phase and NH3 in the lower phase
Anal Bioanal Chem. 2015 Jul;407(18):5503-11. doi: 10.1007/s00216-0158723-1 Overcoming
the equivalent-chain-length rule with pH-zone-refining countercurrent chromatography for the
preparative separation of fatty acids.
Englert M, Vetter W.

J Chromatogr A. 2016 Jan 4;1427:96-101. doi: 10.1016/j.chroma.2015.12.016. Optimisation and establishment of separation conditions of organic acids
from Usnea longissima Ach. by pH-zone-refining counter-current chromatography: Discussion of the eluotropic sequence. Sun C, Liu F, Sun J, Li J, Wang X.

Centrifugal partition extraction in the pH-zone-refining displacement mode: an efficient strategy for the screening and isolation of
biologically active phenolic compounds M Hamzaoui, JH Renault, R Reynaud, J Hubert Journal of Chromatography B 937, 7-12
The fractionation process was performed at a flow rate of 20 mL/min using a biphasic solvent system composed
of terAcWat (4:1:5) in the ascending mode. Sodium hydroxide (40 mM) and trifluoroacetic acid (30 mM) were
used as retainer and displacer agents, respectively.

Fig. 2. pH of the mobile phase, composition and quantities of the fraction pools from PIto PVIII recovered as a function of time during the
pH-zone-refining CPE fractionation process. TTP1 sericic acid; TTP2 trachelosperogenin E; TTP3 sericoside; PP1 gallic acid; PP2
protocatechuic acid; PP3 catechin; PP4 gallocatechin; PP5 epigallocatechin; EAd23,4,3-tri-O-methylflavellagic acid; EAd3 3,3-di-O-
methylellagic acid; EAd4 3,3-di-O-methylellagic acid 4-O-xylopyranoside.
Centrifugal partition extraction in the pH-zone-refining displacement mode: an efficient strategy for the screening and isolation of
biologically active phenolic compounds M Hamzaoui, JH Renault, R Reynaud, J Hubert Journal of Chromatography B 937, 7-12

2. Separation of Bases
C. pH zone refining

Yang, F. Q., Ito, Y., J. Chromatogr. A 2001, 923, 281–285.
Fig. 2. Separation of lappaconitine from a prepurified
extract of A. sinomontanum Nakai. terTetWat (2:2:3,
v/v), 10 mM TEA in the upper organic stationary phase
and 10 mM HCl in the lower phase; sample size: 2.0 g
(A), 6.5 g (B) and 10.5 g (C); flow-rate: 3 ml /min; 860
rpm; Sf: 75.8% (A), 75% (B) and 75.6% (C);.

Fig. 3. crude extract from the Macleaya cordata. :
(A) ChMWat (4:3:3); 10mM HCl in upper aqueous
(stationary) phase and 10mM TEA in lower phase;
(B) ChEMWat (3:1:3:3); 10mM HCl in upper aqueous
phase and 10mM TEA in lower organic phase;
(C) ChEMWat (3:1:3:2); 10mM HCl in upper aqueous
phase and 10mM TEA in lower organic phase;
850 rpm; 2 mL/min: 1.5 g;
Journal of Liquid Chromatography & Related Technologies Volume 38, Issue 20, 2015
Preparative Separation of Chelerythrine and Sanguinarine from Macleaya cordata by pH-Zone-Refining Counter-current Chromatography
DOI:10.1080/10826076.2015.1105257 Qian Liu, Changlei Sun, Fansheng Meng, Wei Zhao, Dapeng Li & Xiao Wang pages 1789-1793

Fig. 12. Separations of a crude alkaloid extract of Crinum moorei obtained by pH-zone-refining CCC with two different
elution modes. (A) Lower aqueous phase mobile and (B) upper organic phase mobile. Experimental conditions: Apparatus
and column: see Fig. 10 caption; solvent system: methyl tert.-butyl ether–acetonitrile–water; stationary phase: (A)
organic phase containing triethylamine at 15 mM and (B) aqueous phase containing HCl at 10 mM; mobile phase: (A)
aqueous phase containing HCl at 5 mM and (B) organic phase containing triethylamine at 10 mM; flow rate: 3.3 ml/min;
sample: 3 g dissolved in 30 ml of each phase; revolution: (A) 800 rpm (600 rpm until 66 ml of the mobile phase was
eluted) and (B) 600 rpm throughout.
Journal of Chromatography A, 1271 (2013) 71– 85 Journal of Chromatography A pH-zone-refining counter-
current chromatography: Origin, mechanism, procedure and applications
Yoichiro Ito

Journal of Chromatography A, 849 (1999) 421–431 Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode Jean-
Hugues Renault*, Jean-Marc Nuzillard, Gae¨lle Le Croue´rour, Philippe The´penier, Monique Ze`ches-Hanrot, Louisette Le Men-Olivier
Fig. 2. (A) Simulated fractogram of a mixture of
vindoline, catharanthine and vincaleukoblastine plus
retaining base (TEA) in the organic
stationary phase. The sample was in the first
theoretical plate. [TEA] 510 mM, [HCl] 58 mM,
[vindoline] 5200 mM, [catharanthine] 5200
mM, [VLB] 5100 mM (concentrations in the first
theoretical plate), number of theoretical plates 5150,
volume of stationary phase 51.5 ml,
volume of aqueous mobile phase 50.25 ml. (B) Purity
and pH profile obtained for the same simulated
separation.

Journal of Chromatography A, 849 (1999) 421–431 Isolation of indole alkaloids from Catharanthus roseus by centrifugal partition chromatography in the pH-zone refining mode Jean-
Hugues Renault*, Jean-Marc Nuzillard, Gae¨lle Le Croue´rour, Philippe The´penier, Monique Ze`ches-Hanrot, Louisette Le Men-Olivier
Fig. 3. UV chromatogram and pH profile
for the separation of vindoline,
catharanthine and vincaleukoblastine.
Sample: ascending mode: 110 mg (0.22
mmol) of vindoline chlorhydrate, 90 mg
(0.24 mmol) of catharanthine
chlorhydrate and 118 mg (0.13 mmol) of
vincaleukoblastine sulfate in 10 ml of
aqueous stationary phase; descending
mode: 100 mg (0.22 mmol) of vindoline,
84 mg (0.25 mmol) of catharanthine and
105 mg (0.13 mmol) of vincaleukoblastine
in 10 ml of organic stationary phase.
terAcWat (TEA, HCl)
4:1:5 (10 mM, 10mM)
http://www.bellybytes.com/herbs/images/periwinkle.jpg

A B
C
A
BC
Fig. 4. pH-Zone-refining counter-current
chromatogram and HPLC control for the
separation of 4.0 g of alkaloid extract from
Nelumbo nucifera leaves. Experiment
condition:
solvent system: petroleum ether–ethyl
acetate–methanol–water (5:5:2:8, v/v/v/v),
10mM TEA in the upper organic
stationary phase and 5mM HCl in the
lower phase;
retention of stationary phase: 60%; flow-
rate: 1.5 mL/min; detection: 254 nm;
revolution speed: 800 rpm.
Fig. 3. High-speed counter-current chromatogram and
HPLC control for the separation of 120mg of alkaloid
extract from Nelumbo nucifera leaves. Experiment
condition:
solvent system: tetrachloromethane–CHCl3–
methanol–0.1MHCl (1:3:3:2, v/v/v/v); retention of
stationary phase: 78%; flow-rate: 1.5 mL/min;
detection: 254 nm; revolution
speed: 800 rpm.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jun 1;878(19):1647-51. doi: 10.1016/j.jchromb.2010.04.020.
Preparative separation of alkaloids from Nelumbo nucifera leaves by conventional and pH-zone-refining counter-current chromatography.
Zheng Z1, Wang M, Wang D, Duan W, Wang X, Zheng C.

Figure 2 1.5 g of alkaloid extract
from Nelumbo nucifera:
terWat, 10mM TEA in the upper
organic stationary phase and
5mM HCl in the lower phase; Sf
– 78%; flow-rate – 1.5mL/min;
800 rpm.
1.5 g of alkaloid extract from
N. nucifera HEMWat (5:5:5:5), 10mM TEA in the upper organic
stationary phase and 5mM HCl in the lower phase; Sf – 70%; flow-rate
– 1.5 mL/min; 800 rpm.
1.5 g of alkaloid extract from N.
nucifera HEMWat (5:5:2:8),
10mM TEA in the upper organic
stationary phase and 5mM HCl in
the lower phase; Sf – 57%; flow-
rate – 1.5 mL/ min; 800 rpm.
J Sep Sci. 2010 Mar;33(4-5):539-44. doi: 10.1002/jssc.200900561. Preparative separation of alkaloids from
Nelumbo nucifera Gaertn by pH-zone-refining counter-current chromatography.
Wang X1, Liu J, Geng Y, Wang D, Dong H, Zhang T.

Fig. 3. Dactylicapnos scandens. Column
volume : 250 mL; PetEMWat (3:7:1:9,
v/v), 20 mM TEA in the upper organic
stationary phase and 5 mM HCl in the
lower phase; Sf 60%;: 1.5 mL/min;
detection wavelength: 254 nm; 750 rpm;:
1.0 g of crude alkaloids.
Fig. 4. D. scandens. column
volume 500 mL; PetEMWat
(3:7:1:9, v/v), 20 mM TEA in the
upper organic stationary phase
and 5 mM HCl in the lower
phase; Sf: 60%; 1.5 mL/min;
detection wavelength: 254 nm; :
750 rpm: 1.0 g of crude alkaloids.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Dec 1;879(31):3767-70. doi: 10.1016/j.jchromb.2011.10.013. Preparative isolation of alkaloids
from Dactylicapnos scandens using pH-zone-refining counter-current chromatography by changing the length of the separation column. Wang X, Dong
H, Yang B, Liu D, Duan W, Huang L.

B D C
Conventional HSCCC separation of fraction II (Fig. 2). Solvent system: HEMWat (7:3:6:4, v/v); revolution speed: 800
rpm; flow rate: 1.5 mL/min; sample size: 150 mg; UV detection wavelength; Sf: 80%.
pH-zone-refining crude
alkaloids from Stephania
kwangsiensis. HEMWat
(3:7:1:9, v/v); 10 mmol/L
TEA in upper organic phase,
5 mmol/L HCl in lower
aqueous phase: 800 rpm; 1.5
mL/min; sample size: 2.0 g;
A B + C + D
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Apr 15;879(13-14):945-9. doi: 10.1016/j.jchromb.2011.02.051.
Combinative application of pH-zone-refining and conventional high-speed counter-current chromatography for preparative separation of alkaloids from Stephania kwangsiensis.
Dong H1, Zhang Y, Fang L, Duan W, Wang X, Huang L.

Large-scale separation of alkaloids from Gelsemium elegans by pH-zone-refining counter-current chromatography with a new solvent system
screening method Journal of Chromatography A, Volume 1307, 13 September 2013, Pages 80-85 Lei Fang, Jie Zhou, YunLiang Lin, Xiao Wang,
Qinglei Sun, Jia-Lian Li, Luqi Huang
pH-zone-refining CCC separation of alkaloids from G.
elegans.: HEMWat, 10 mM triethylamine in upper
organic phase, 10 mM hydrochloric acid in lower
aqueous phase, (A) (5:5:5:5, v/v), (B) (5:5:3:7, v/v), (C)
(5:5:1:9, v/v), and (D) (3:7:1:9, v/v); 800 rpm; 2.0
mL/min; sample size: (A) 1.0 g, (B) 1.5 g, (C) 3.1 g, and
(D) 4.5 g; stationary phase retention: (A) 59%, (B) 58%,
(C) 50%, and (D) 47%;

Fig. 2. pH-zone-refining CCC separation of G. elegans extract. :
MtBE/CH3CN/water (3:1.5:4, v/v), 20mMTEA in the upper organic
stationary phase and 10mM HCl in the lower aqueous phase; sample
size: 1.0 g (A) and 1.5 g (B); flow-rate: 2ml/min; detection: 254 nm;
revolution speed: 850 rpm; retention of stationary phase: 58.8% (A)
and 58.3% (B).
J Chromatogr A. 2011 Jun 10;1218(23):3695-8. doi: 10.1016/j.chroma.2011.04.025. Preparative separation of alkaloids from Gelsemium elegans
Benth. using pH-zone-refining counter-current chromatography. Su YP1, Shen J, Xu Y, Zheng M, Yu CX.

3. Ion Exchange
C. pH zone refining

strong ion-exchange displacement centrifugal partition chromatography (SIXCPC).
Journal of Chromatography A, 1170 (2007) 44–51 Strong ion-exchange centrifugal partition chromatography as an efficient method for the large-
scale purification of glucosinolates Alix Toribio, Jean-Marc Nuzillard, Jean-Hugues Renault ∗
EBuWat (Aliquat 336, NaI) 3:2:5 (160 mM, 80 mM)
https://upload.wikimedia.org/wikipedia/commons/thumb/1/19/Sinalbin.svg/2000px-Sinalbin.svg.png
C.3. ion-exchange displacement

Journal of Chromatography A, 1170 (2007) 44–51 Strong ion-exchange centrifugal partition chromatography as an efficient method for the large-
scale purification of glucosinolates Alix Toribio, Jean-Marc Nuzillard, Jean-Hugues Renault ∗
The column was filled with organic stationary
phase (SP) in the descending mode, at 30
mL/min and with a 300 rpm column rotation
speed. The sample was then injected at 2
mL/min at 1200 rpm. NaI-free mobile phase
(MP) (314 and 80mL for broccoli extract and
white mustard extract, respectively) was
pumped at 2 mL/min in order to allow
extraction
of the GSLs into the stationary phase and
elution of polar impurities such as polyphenols
or free sugars (40 min for S.
alba extract and 157 min for B. oleracea
extract). The displacing aqueous mobile phase
was then pumped at 2 mL/min, and the
fractions were collected every minute.

Pilot-scale ion-exchange centrifugal partition chromatography: Purification of sinalbin from white mustard seeds Journal of Separation
Science. Volume 32, Issue 11, 2009 Pages 1801–1807 Alix Toribio, Jean-Marc Nuzillard, Benoît Pinel, Leslie Boudesocque, Michel Lafosse,
François De La Poype, Jean-Hugues Renault
http://www.apnfs.info/wp-content/uploads/2015/08/mustard-seeds-yellow-1.jpg

Preparative isolation of glucosinolates from various edible plants by strong ion-exchange centrifugal partition chromatography. Separation and Purification Technology, v.83, 2011 Nov 15,
p.15(8) Toribio, Alix Boudesocque, Leslie Richard, Bernard Nuzillard, Jean-Marc Renault, Jean-Hugues
C.3. ion-
exchange
displacement

Preparative isolation of glucosinolates from various edible plants by strong ion-exchange centrifugal partition chromatography. Separation and Purification Technology, v.83, 2011 Nov 15,
p.15(8) Toribio, Alix Boudesocque, Leslie Richard, Bernard Nuzillard, Jean-Marc Renault, Jean-Hugues

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