Optimization parameters in Countercurrent Chromatography

Optimization
https://photos.prnewswire.com/prnvar/20160223/336432

Sample Preparation
Equal amounts of upper and lower phases.
(Usually best for overall solubility)
Add to sample loop in alternating plugs
Leave a buffer of mobile phase on each side of the sample loop
Need to filter insoluble material?

Column Loading
http://www.microcontractor.org/uploads/4/4/9/7/44977385/8245406_orig.jpg
1. Column loading
- higher sample concentration
- more sample volume
2. scale-up

Preparative loading of CCS instruments
322 articles
52 journals
2008 through 2012.
15
52
27
24
4
0
10
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30
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50
60
< 100 101-500 501-1000 1001-5000 > 5000
number of separations
mg sample
52
133
53 49
17
0
20
40
60
80
100
120
140
< 100 101-500 501-1000 1001-5000 > 5000
mg sample
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.
Pauli, G. F.; Pro, S. M.; Friesen, J. B., Countercurrent
separation of natural products. Journal of Natural Products
2008, 71, 1489-1508.
2000 through 2007 2008 through 2012
137 articles
24 journals
2000 through 2007.
Column Loading

Column Loading
15
40
61
102
15
9
0.01 - 0.20
0.21 - 0.50
0.51 - 1.00
1.01 - 5.0
5.2 - 10.0
10.5 - 21.0
mg sample /mL column volume
Column Loading: HSCCC instruments
0.01 - 0.20
0.21 -0.50
0.51 - 1.00
1.01 - 5.0
5.2 - 10.0
10.5 - 375
B
21
26
129
66
44
18
Column Loading: All CS instruments
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.

Column Loading
0
2000
4000
6000
8000
10000
12000
14000
16000
0 50000 100000 150000 200000 250000
v
o
l
u
m
e
m
L
mg loaded
mg loaded & volume
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v
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mg loaded
mg loaded & volume

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

GUESSmix Used to Evaluate SSs
The distribution coefficient (K) is a constant for a particular substance
in a particular solvent system.
Independent of: - column volume
- instrument model
- normal or reverse-phase mode
- run time
- rotation speed
- flow rate
G Mix H/tBME/ACN/Water 4:6:5:5 10/12/06
-0.1
0.8
0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375
mL & mn
A
280nm
230nm
K-Based Chromatography

CCC Chromatogram
0
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700
A254
mL
GUESSmix in ChMWat 10:7:3 12/14/12, 35 degrees N Phase
Compounds were identified using TLC and CCC peaks
Q HD
FUE
C
V
r

HPLC
• Next, HPLC was used to further identify compounds along with the
TLC plates and GC chromatograms
-56.05
43.95
143.95
243.95
343.95
443.95
543.95
643.95
743.95
843.95
943.95
1043.95
1143.95
1243.95
1343.95
1443.95
0 5 10 15 20 25 30 35 40 45 50 55 60 65
X
T
Gr
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72
A254
tube
GUESSmixin DMWat10:6:4 11/02/12, 35degreesN Phase
G
T
r
X
HPLC of test tube 69
69
69

Emphasize the importance of K
Using GUESSmix to explore solvent system families.
Friesen, J.B. Pauli, G.F. Analytical Chemistry 79: 2320-2324 (2007)
Symmetry
Midline
0
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
A
M
Q
V
U
F
N
Z E

Column Loading
What is the effect of column loading on
K value and resolution?

10mg/compound
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
5 mg/compound
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
20mg/compound
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
2.29 2.67 3.2 4.00 5.33 8.00 16
8
Sf = 0.52
Sf = 0.54
Sf = 0.56
Column Loading

30mg/compound
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
50mg/compound
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
40mg/compund
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
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
Sf = 0.59
Sf = 0.59
Sf = 0.44
Column Loading

Column Loading
Solute-solute interactions may decrease or
increase resolution.
Solute-solute interactions may affect the
apparent (experimental) K value.

Fig. 3. APCI-MS (pos. mode) molecular weight profile
chromatogram generated by sequential injections of even
numbered fractions of the HPCCC preparative separations of S.
terebinthifolius berries on the Midi and selected ion traces of
peaks 1–3. Each signal contained the complete APCI-MS profile
information of compounds present in a single assay tube (distance
between two monitoring signals is equivalent to 50.0 mL – Midi I
to III and 80 mL – Midi IV).
J Chromatogr A. 2015 Apr 10;1389:39-48. doi: 10.1016/j.chroma.2015.02.005.
Schinus terebinthifolius scale-up countercurrent chromatography (Part I): High
performance countercurrent chromatography fractionation of triterpene acids with off-
line detection using atmospheric pressure chemical ionization mass spectrometry. Vieira
MN, Costa Fd, Leitão GG, Garrard I, Hewitson P, Ignatova S, Winterhalter P, Jerz G
Column Loading
n-heptane/ethyl acetate/methanol/water (6:1:6:1)

Figure 4. HSCCC chromatogram of crude flavonol glycosides from Ginkgo biloba
leaves at flow rate 1.2 mL/min. n-hexane/butanol/ethyl acetate/methanol/0.5%
acetic acid (2:1:7:2:8)
J. Sep. Sci. 2007, 30, 2153 – 2159
Qiang Zhang, Li-Juan Chen, Hao-Yu Ye, Lei Gao, Wenli Hou, Minghai Tang, Guangli Yang, Zhenhua Zhong,
Yuan Yuan, Aihua Peng, Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves
by high-speed counter-current chromatography
Column Loading

(a) HSCCC chromatogram when injection volume was 100 mg for MD-R.
(b) HSCCC chromatogram when injection volume was 600 mg for MD-R.
HEMWat 10:2:5:7, 800 rpm, lower phase mobile 2 mL/mi. 280 nm TBE300
J Chromatogr B 2016 Feb 1;1011:99-107. doi: 10.1016/j.jchromb.2015.12.051.
Separation and preparation of 6-gingerol from molecular distillation residue of Yunnan ginger rhizomes by high-speed counter-current
chromatography and the antioxidant activity of ginger oils in vitro. Gan Z, Liang Z, Chen X, Wen X, Wang Y, Li M, Ni Y.
Column Loading

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.
Figure 5. HSCCC chromatograms of the EPS at different load masses. HSCCC conditions: solvent system: EBuWat (9:1:10);
revolution speed: 900 rpm; separation temperature: 30 °C; flow rate: 2.2mL/min; detection wavelength: 280 nm; sample size:
(A) 50mg, (B) 100mg, (C) 150mg, (D) 200mg of the EPS in 5mL of the upper phase and 5 mL of the lower phase.
Column Loading

Scale-Up
http://ian.umces.edu/imagelibrary/albums/userpics/12789/normal_ian-symbol-mountains-snowcaps-and-foothills.png

Fig. 6. Comparison of chromatograms for different coils operated in similar conditions with their corresponding parameters in the reversed-phase
mode. (A) The chromatogram for the 4.7 ml coil on Mini-DE: HepEMWat phase system of 2:3:2:3; rotation speed, 2000 rpm; sample loop, 0.1ml;
sample concentration, 20 mg/ml; flow rate, 1.0ml/min; temperature, 25 ◦C; Sf = 53.03%. (B) The chromatogram for the 17.2 ml coil on Mini-DE:
HepEMWat phase system of 2:3:2:3; rotation speed, 2000 rpm; sample loop, 0.43 ml; sample concentration, 20 mg/ml; flow rate, 1.0ml/min;
temperature, 25 ◦C: Sf = 53.01%. (C) The chromatogram for the 915.5 ml coil on Midi-DE: HepEMWat phase system of 2:3:2:3; rotation speed,
1250 rpm; sample loop, 20.0 ml; sample concentration, 20 mg/ml; flowrate, 50.0 ml/min; temperature, 25 ◦C; Sf = 53.33%.
Journal of Chromatography A, 1194 (2008) 192–198 How to realize the linear scale-up process for rapid purification using high-performance counter-current
chromatography Yuan Yuan, BiqinWang, Lijuan Chen, Houding Luo, Derek Fisher, Ian A. Sutherland,Yuquan Wei
Scale-up

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
Scale-up
Fig. 1. 4.6 L Maxi separation of benzyl alcohol and p-cresol scaled up 850× from the Milli-CCC separation (inset). Operating conditions for 4.6 L Maxi: speed
600 rpm, flow 850 mL/min; sample load, 290mL (6.3% Vc) 12.2 g BA, 5.8 g PC. Run conditions for the Milli-CCC: speed 2100 rpm, flow 1 mL/min; sample
loading condition the same concentration and proportion of column volume as for Maxi. Phase system: HEMWat (14:1:5:10, v/v/v/v). Sf before injection
80.0%, after separation 40.8% for the Maxi run.

Purity values and centrifugal partition
chromatography elution profile of the major
compounds isolated from a crude aqueous
extract of Stevia leaves using the ASCPC250®
instrument (experiment A, 500 mg injected;
experiment B, 1 g injected) or the FCPE300®
instrument (experiment C, 5 g injected).
Sf = 0.75, 0.70 & 0.70 for A, B & C.
1200 rpm A & B, 1000 rpm C.
10 mL/min A & B, 20 mL/min C.
Planta Med. 2015 Nov;81(17):1614-20. doi: 10.1055/s-0035-1545840. Intensified Separation of Steviol Glycosides from
a Crude Aqueous Extract of Stevia rebaudiana Leaves Using Centrifugal Partition Chromatography. Hubert J, Borie N,
Chollet S, Perret J, Barbet-Massin C, Berger M, Daydé J, Renault JH.
Scale-up

Figure 3.2 The importance of the amount of stationary phase retained in
a 100 ml CCC column (vertical dotted line). Chromatograms of the same
mixture of seven solutes (listed in Table 3.1) obtained with the same CCC
hydrodynamic column and the same biphasic liquid system: hexane/ethyl
acetate/methanol/water 4:6:4:6 v/v. Aqueous lower mobile phase, 2 ml/
min, rotor rotation 900 rpm, average efficiency ∼300 plates
Separations with a Liquid Stationary Phase: Countercurrent Chromatography or Centrifugal Partition
Chromatography Alain Berthod and Karine Faure
Analytical Separation Science, First Edition. Edited by Jared L. Anderson, Alain Berthod,
Verónica Pino Estévez, and Apryll M. Stalcup.
2015 Wiley-VCH Verlag GmbH & Co. KGaA.
Sf

6 5
16
42
68
73
44
26
2
0
10
20
30
40
50
60
70
80
10-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100
reported Sf values
Sf

Determination of Sf
• (i) the carry-over method Sf(CO): this approach measures, in a graduated cylinder, the
amount of stationary phase carried over as the column is equilibrated with the
mobile phase. The amount of stationary phase displaced is called the “carry over
volume” or V(CO). In the case of large volume columns, V(CO) is considered to be
equal to the mobile phase volume inside the column (VM).
• (ii) void volume determination by UV detection Sf(UV): this method is routinely
employed with crude natural extracts, is to identify the mobile phase front by
unretained UV-active sample components that are almost always present in
complex natural mixtures. The void volume (V(UV)) is determined by taking the
analyte retention volume (VR) of an unretained component(s) (VR
0) to be equal to
VM.
• (iii) volumetrics of extruded mobile phase Sf(MP): the third method is based on the
determination of VM by measuring the volume of mobile phase extruded (V(MP))
from the column after the separation is complete.

Flow Rate
https://farm7.staticflickr.com/6081/6077573063_2bdf56e975_z.jpg

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 (constant rpm)
Friesen JB, Pauli GF GUESSmix-guided optimization of elution–extrusion counter-current separations. Journal of Chromatography A 1216: 4225-4231 (2009)

Sf and Flow Rate
Fig. 3. Stationary (upper) phase retention ratio in percentage of the column volume plotted versus the square root of the lower
mobile phase flow rate. CCC column volumes and rotor rotations: Mini 1.6mm I.D. 20.8mL and 1800 rpm; Mini 0.8mm I.D.
19.5mL and 2100 rpm; SFCC 1-coil 54mL and 800 rpm; SFCC 3-coil 156mL and 800 rpm. HepEMWat 2:3:2:3, head to tail
flowing direction. The regression equations give A intercepts and B slopes (Eq. (5)). The R2 regression coefficient is listed
below its equation.
Berthod2009_JCA_1216_4169_SmallVolume

CPC
Fig. 4. Evolution of the stationary phase (Sf) retention as a function of flow rate (F) for different rotational
speeds; Sf0, y-abscises intercept, corresponds to the cells fraction of the column volume. Dots are experimental
data; straight lines show tendency and flooding transition
J Chromatogr A. 2015 Apr 24;1391:80-7. doi: 10.1016/j.chroma.2015.03.005. Modeling pH-zone refining countercurrent chromatography: a
dynamic approach. Kotland A, Chollet S, Autret JM, Diard C, Marchal L, Renault JH.
Sf and Flow Rate

Flow Rate
Fig. 3. (a–c) Separation of the GUESS mixture as described in [15] using DE
Spectrum preparative coils at 80×g (918 rpm), 136mL column volume,
HEMWat (2:3:2:3), lower layer as mobile phase, 1.5, 3 and 6mL/min, head to
tail elution, Sf = 77 % (1.5mL/min), 64% (3 mL/min) and 53% (mL/min); 60mg
total loading (5mg of each 12 standards: aspirin (A), -carotene, caffeine (C),
nicotinic acid (D), estradiol, ferulic acid (F), naringenin, carvone, red new
Coccine (R), quercetin (Q), umbelliferone (U), vanillin (V)), monitored by DAD
254 nm, compared separation times 130, 60 and 32 min, respectively.
1.5 mL/min
Sf = 0.77
3 mL/min, Sf = 0.64
6 mL/min
Sf = 0.64
918 rpm
Performance comparison using the GUESS mixture to evaluate counter-current chromatography
instruments Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4181-4186 Hacer
Guzlek, Philip Leslie Wood, Lee Janaway

Flow Rate
Fig. 4. Separation of the GUESS mixture using DE Spectrum preparative coils at
243×g (1600 rpm), 136mL column volume, HEMWat (2:3:2:3), lower layer as
mobile phase, 1.5, 3 and 6 mL/min, head to tail elution, Sf = 88% (1.5 mL/min),
85% (3 mL/min) and 73% (6 mL/min); 60mg total loading for 1.5 and 6mL/min;
total loading for 3 mL/min was 55mg because red new Coccine was not available
(5mg of each 12 standards: aspirin (A), -carotene, Caffeine (C), nicotinic acid (D),
estradiol, ferulic acid (F), naringenin, carvone, red new Coccine (R), quercetin
(Q), umbelliferone (U), vanillin (V)), monitored by DAD 254 nm, compared
separation times 105, 60 and 30 min, respectively.
1600 rpm
1.5 mL/min
Sf = 0.88
3 mL/min, Sf = 0.85
6 mL/min
Sf = 0.73
Performance comparison using the GUESS mixture to evaluate counter-current chromatography
instruments Journal of Chromatography A, Volume 1216, Issue 19, 8 May 2009, Pages 4181-4186
Hacer Guzlek, Philip Leslie Wood, Lee Janaway

Figure 3. HSCCC chromatogram of crude flavonol glycosides from Ginkgo biloba leaves. HSCCC conditions: column volume: 40 mL;
solvent system: hexane/butanol/ethyl acetate/methanol–0.5% acetic acid (1:0.5:3.5:1:4); stationary phase: lower; 1600 rpm; detection
wavelength: 254 nm; temperature: 25 oC; sample concentration: 20 mg/mL; Sf phase at 1.0, 1.2, and 1.5 mL/min flow rates: 78.1, 60.1,
and 45.4%, respectively.
J. Sep. Sci. 2007, 30, 2153 – 2159
Qiang Zhang, Li-Juan Chen, Hao-Yu Ye, Lei Gao, Wenli Hou, Minghai Tang, Guangli Yang, Zhenhua Zhong,
Yuan Yuan, Aihua Peng, Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves
by high-speed counter-current chromatography
Flow Rate

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.
Figure 4. HSCCC chromatograms of the EPS at different flow rates. HSCCC conditions: EBuWat (9:1:10): 900 rpm: 30
°C;: 50mg of the EPS in 5mL of the upper phase and 5mL of the lower phase; detection 280nm; flow rate: (A)
1.2mL/min, (B) 1.5mL/min, (C) 1.8mL/min, (D) 2.2mL/min.
Flow Rate Gradients

Figure 3. HPCCC separation patterns of the acetone-soluble extract of fermented C.
sinensis leaves using HEMWat (1:9:1:9, v/v) system with rotational speed at 1600
rpm. (A) flow-rate at 3.0 mL/min; (B) flow-rate at 5.0 mL/min; (C) flow-rate at 8.0
mL/min; (D) gradient flow-rate at 3.0 mL/min in 0–45 min, and 5.0 mL/min in 45–
130 min. Peaks 1: caffeine, 2: (−)-epigallocatechin 3-O-gallate, 3: (−)-gallocatechin 3-
O-gallate, and 4: (−)-epicatechin 3-O-gallate.
Molecules 2015, 20, 13216-13225; doi:10.3390/molecules200713216
Separation of Polyphenols and Caffeine from the Acetone Extract of Fermented Tea Leaves (Camellia sinensis) Using High-Performance Countercurrent
Chromatography Soo Jung Choi, Yong Deog Hong, Bumjin Lee, Jun Seong Park 2, Hyun Woo Jeong, Wan Gi Kim, Song Seok Shin and Kee Dong Yoon
Flow Rate Gradients

Fig. 3. HSCCC chromatograms of the EFS. Peak 1: procyanidin B3, Peak 2: procyanidin B1, Peak 3: catechin, and Peak 4:
procyanidin B7. HSCCC conditions: solvent system: HEMWat (0.75:12.5:1:12.5, v/v/v/v); stationary phase: upper phase: 900
rpm; 30 C; 300 mg of the EFS: 280 nm; flow rate: 0–100 min, 1.5 mL/min, ∼100 min, (A) 1.5 mL/min, (B) 2.0 mL/min, (C) 2.5
mL/min, (D) 3.0 mL/min. Rs of 1.5 mL/min:67.19%; 2.0 mL/min:65.94%; 2.5 mL/min:64.06%; 3.0 mL/min:62.50%.
Journal of Liquid Chromatography & Related Technologies, 38: 1486–1493, 2015
DOI: 10.1080/10826076.2015.1063506 Separation and Purification of Four Flavan-3-ols From Iris
Lactea Pall. var. Chinensis (Fisch.) Koidz by High-Speed Counter-Current Chromatography with Flow-Rate Gradient
HUANHUAN LV, JIAN OUYANG, XIAOYAN WANG, XIAOFENG MA,2YOURUI SUO, and HONGLUN WANG
Flow Rate Gradients

Flow Rate & rpm
0
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r
p
m
flow mL/min
flow rate & rpm
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0 1 2 3 4 5 6
r
p
m
flow mL/min
flow rate & rpm
Countercurrent Separation of Natural Products: An Update.
Journal of Natural Products 2015, 78, 1765-1796.

Flow Rate & Volume
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flow rate & volume
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0 20 40 60 80 100 120 140 160
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o
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e
flow mL/min
flow rate & volume

Flow Rate/Temperature/Purity
Figure 5. Response surface plots for the optimization of HSCCC process. A) Effect of temperature and
flow rate on purity of rutin at a constant revolution speed of 850 rpm;
Chem. Ind. Chem. Eng. Q. 21 (2) 331−341 (2015) PREPARATIVE ISOLATION AND PURIFICATION OF SEVEN COMPOUNDS FROM Hibiscus mutabilis L. LEAVES
BY TWO-STEP HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY ZHUONI HOU, XIANRUI LIAN, FENG SU, WEIKE SU,
Figure 3. HSCCC Chromatograms of the first separation. HSCCC
Conditions: solvent system: EBuWat (6:1:9 volume ratio); stationary
phase: upper phase. Flow rate: 1.11 mL/min, revolution speed: 800
rpm, temperature: 30 °C; stationary phase: upper organic phase;
detection wavelength: 254 nm; sample size: 100 mg.

Figure 5. Response surface plots for the optimization of HSCCC process. B) influence of revolution
speed and temperature on purity of rutin at a definite flow rate of
1.5 mL/min;
rpm/Temperature/Purity

Figure 5. Response surface plots for the optimization of HSCCC process. C) interaction between
revolution speed and flow rate at a fixed temperature of 25 °C.
Flow Rate/rpm/Purity

0
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rpm and volume
rpm & Volume
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v
o
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e
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rpm and volume

Flow Rate & rpm
How does the interplay of flow rate and rpm affect K
values and resolution?
600 rpm
1.5 mL/mn
800 rpm
1.5 mL/mn
1000 rpm
1.5 mL/mn
600 rpm
2 mL/mn
800 rpm
2 mL/mn
1000 rpm
2 mL/mn
600 rpm
3 mL/mn
800 rpm
3 mL/mn
1000 rpm
3 mL/mn

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

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
800 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 = 0.36
Sf = 0.48
1000 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 = 0.55
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 rpm

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
Sf = 0.52
2.29 2.67 3.2 4.00 5.33 8.00 16
8
Increasing Flow Rate & rpm
800 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
Sf = 0.54
2.29 2.67 3.2 4.00 5.33 8.00 16
8
1000 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 = 0.55
2.29 2.67 3.2 4.00 5.33 8.00 16
8

Flow Rate & rpm
Increasing flow rate increases
apparent (experimental) K
values and decreases
resolution.
Especially at lower rpm.
Increasing rpm decreases apparent
(experimental) K values and increases
resolution. Especially at higher rpm.
K
flow rate rpm
Sf

Temperature
How does temperature affect the K value and
resolution?
Tauto
TBE300A
temperature of CS experiments
0
5
10
15
20
20 25 30 35
C
numberofarticles
38 data points

15 C
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
5 C
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
10 C
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 = 0.45
Sf = 0.45
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
Sf = 0.45
2.29 2.67 3.2 4.00 5.33 8.00 16
8
Temperature

20 C
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
25 C
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 = 0.50
Sf = 0.54
15 C
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 = 0.45
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
Temperature

30 C
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
35 C
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
25 C
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 = 0.53
Sf = 0.57
Sf = 0.54
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
Temperature

no temperature regulation
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
25 C
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
30 C
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
Sf = 0.54
Sf = 0.53
Sf = 0.53
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
Temperature

Temperature
Temperature influences both
K values and resolution.
Generally, K decreases while Temperature
increases.
Generally, Temperature influences
resolution because compounds respond
differently to Temperature changes.
Sf as a function of Temperature
0.4
0.5
0.6
0 10 20 30 40degrees C
Sf

Temperature
Generally, K tends toward unity while
Temperature increases.
K Value as a Function of Temperature
0
2
4
6
8
10
1 2 3 4 5 6 7 8
temperature
K
F
U
V
Q
M
N
E
5 10 15 20 25 30 35room

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.
Temperature Effects
Table 2
Partitioning coefficients K of α-carotene and β-
carotene and separation factor between them at
different temperatures with the two-phase
solvent system H/benzotrifluoride/Ac
(10:3.5:6.5, v/v/v) determined by HPLC/UV–vis

Parameter Type Parameter Experimental report
Essential Important Optional
Operational Flow rate E
Rpm E
Solvent system solvent and volume ratios E
Mobile phase identity E
Flow direction (head-to-tail, tail-to-head) E
Stationary phase volume ratio (Sf) E
Switch volume (Vex) of elution extrusion if used E
Column equilibration and sample injection method I
Temperature I
Pressure variation during experiment O
Gravitational field generated by rotation O
Solvent system phase composition O
SS interfacial tension O
SS density difference of phases O
Viscosity of each phase O
pH of aqueous phase O
Sample Loading mass of sample E
Loading volume E
Recovery mass of individual compounds I
Enrichment I
Composition of active fractions and analytical method I
Purity of target analytes and determination method I
Partition coefficient (K) of target analytes I
Percent recovery of target analytes O
Pauli GF, Pro S, Friesen B Countercurrent Separation of Natural Products Journal of Natural Products 71: 1489-1508 (2008)
dx.doi.org/10.1021/np800144q
Reporting Operational Parameters

Reporting of Separation Parameters:
Process Throughput (PT in Grams of Sample Processed per Hour of Separation Time),
Process Efficiency (PE in Grams of Sample Processed per Hour of Separation Time),
Process Environmental Risk Factor (ER in Liters of Solvent per Gram of Product),
Process' General Evaluation Factor (GE in Grams of Sample Times Grams of Product per Hour of
Separation Time per Liter of Solvent)
natural
product(s)
(no. of cpds)
source solvent system PT [g/h] PE [g/h] ER [l/g] GE [g2
/(h·l)] ref
ginsenosides
(4)
Panax ginseng
roots
DiMWat and
HBuWat (0.1%
formic acid)
5.14 0.21–0.54 0.065–0.18 1.17–8.27 140
salvianolic
acid B
Salvia miltiorrhiza
rhizomes
HEMWat (0.1%
acetic acid)
2.23 0.77 4.0 0.192 250
ginsenosides
(4)
Panax ginseng
roots
DiIsoWat
(ammonium
acetate)
0.23 6.73–14.6 0.06–0.71 9.5–243 251
geniposide Gardenia
jasminoides fruits
EBuWat 5.0 0.55 4.9 0.113 252
(140) Cheng, Y. J.; Zhang, M.; Liang, Q. L.; Hu, P.; Wang, Y. M.; Jun, F. W.; Luo, G. A. Sep. Purif. Technol. 2011, 77, 347-354.
(250) Zhang, M.; Ignatova, S.; Liang, Q. L.; Jun, F. W.; Sutherland, I.; Wang, Y. M.; Luo, G. A. J. Chromatogr. A 2009, 1216, 3869-3873.
(251) Qi, X. C.; Ignatova, S.; Luo, G. A.; Liang, Q. L.; Jun, F. W.; Wang, Y. M.; Sutherland, I. J. Chromatogr. A 2010, 1217, 1995-2001.
(252) Zhang, M.; Ignatova, S.; Hu, P.; Liang, Q. L.; Wang, Y. M.; Sutherland, I.; Jun, F. W.; Luo, G. A. Sep. Purif. Technol. 2012, 89, 193-198.

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