1.
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.
CounterCurrent Separation
Introduction to Instrumentation
§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

3.
Liquid/Liquid Separation (LLS)
Kuhni
Extraction
Columns
Continuous
Mixer-Settler
Liquid-Liquid
Extraction
Liquid/Liquid Chromatography
A. Martin & R. Synge
Centrifugal
Countercurrent
Separation (CCCS)
Gravitational
Countercurrent
Separation (GCCS)
Countercurrent
Chromatography
(CCC)
“hydrodynamic”
Centrifugal Partition
Chromatography
(CPC)
“hydrostatic”
Droplet
Counter
Current
Chromatogr.
Craig
Counter
Current
Distribution
Kostanyan
Pulsed
Rotational
Locular
Separatory
Funnels
Countercurrent Separation (CCS)
Friesen JB, McAlpine JB, Chen SN, Pauli GF
Countercurrent Separation of Natural Products: An Update
Journal of Natural Products 78: 1765-1796 (2015)
dx.doi.org/10.1021/np501065h
CCS Cladistic Tree

4.
Partition coefficient (K)
Concentration of analyte in one phase/
Concentration of analyte in the other phase
shake flask (partitioning) experiment
= 4/4 = 1
= 12/36 = 1/3
K = Cupper/Clower

5.
Countercurrent Distribution
Series of mixed and equilibrated individual cells
All cells contain stationary phase (lower phase in CCD instruments) at the beginning.
The first cell is mixed and allowed to settle.
The mobile phase is passed to the next cell in the series.
New mobile phase is added to the first cell.
The first cell contains an equal amount of upper and lower phase
plus the sample at the beginning.

6.
2
3
2
9
2
9
2
27
4
12
4
36
separate phases
equilibrate
1
2

7.
1
1
1
2
2
6
2
12
1½
7
1½
14
1
9
1
18
½
6
½
12
1½
2.7
1½
5.3
½
0.7
½
0.3
4
5

8.
Countercurrent Distribution
Lyman C. Craig
Sequential countercurrent extractions can separate solutes
with only small differences in K. However, the technique, if
performed with separatory funnels, is quite tedious.
In 1944 Lyman C. Craig developed a device to automate
countercurrent distribution. This device used a series of
glass vessels.
8
http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1951.tb48879.x/pdf

9.
CCD apparatus takes advantage of
successive extractions
Countercurrent distribution
Jensen 2010, Museum Notes p. 1 Craig CCD Train
http://www.erittenhouse.org/wp-content/uploads/2015/07/Fig.-5rs21-973x1030.jpg

10.
CCD output
Isopropyl ether and phosphate buffer pH 4.93
http://www.jbc.org/content/174/1/221.full.pdf

11.
CounterCurrent Distribution
CCD equations:
The rate of migration of the band of a homogeneous substance, that is the position of
its maximum, can be calculated from Equation 1 at any time during operation, after a
certain number of plates, n, have been applied
IDENTIFICATION OF SMALL AMOUNTS OF ORGANIC COMPOUNDS BY DISTRIBUTION
STUDIES III. THE USE OF BUFFERS IN COUNTER-CURRENT DISTRIBUTION.
BY LYMAN C. CRAIG, CALVIN GOLUMBIC, HAROLD MIGHTON, AND ELWOOD TITUS
Journal of Biological Chemistry, 1945, vol. 161, p. 321

12.
http://www.chem.uoa.gr/Applets/AppletCraig/Appl_Craig2.htm 12

13.
Droplet Countercurrent Chromatography
http://images.all-free-download.com/images/graphicthumb/different_shapes_water_drop_creative_design_546322.jpg

14.
Droplet Countercurrent Chromatography
Eyela DCCC

15.
Droplet Countercurrent Chromatography
Ascending mode: lower phase is held
stationary in tubes and upper phase
bubbled from the bottom.
Descending mode: upper phase is held
stationary in tubes and lower phase
bubbled from the top.
continuous flow

16.
Journal of Chromatography A
Volume 128, 1976, Pages 218-223
Droplet counter-current chromatography for the separation of plant products
Yukio Ogihara, Osamu Inoue Hideaki Otsuka, Ken-Ichi Kawai 1, Takenori Tanimura, Shoji Shibata
Droplet Countercurrent Chromatography

17.
Hostettmann, 1980 Droplet Counter-Current Chromatography and its Application to the Preparative Scale Separation of Natural Products 39, p. 1
Droplet Countercurrent Chromatography

18.
Head
Tail
Correct
CorrectX
Descending
Mode
Lower
Phase
Mobile
Ascending
Mode
Upper
Phase
Mobile
X
http://dingo.care2.com/pictures/greenliving/uploads/2013/10/cat-tail.jpg
https://cdn.shopify.com/s/files/1/0224/1915/products/grey-tabby-kitty-cat-head-shaped-vinyl-animal-photo-print-clutch-bag-dotoly.jpg?v=1398690617

19.
Centrifugal Partition Chromatography

20.
http://www.labx.co.kr/mall/images/Fg21Dr_FC.gif
Centrifugal
Partition
Chromatography

21.
Centrifuge is used to hold
one phase stationary.
cells or chambers
reminiscent of CCD
vessels
http://www.labx.co.kr/mall/images/Fg2Dr_FC.gif
Centrifugal Partition Chromatography

22.
(High Speed)
Countercurrent Chromatography

23.
Continuous Countercurrent
Separation in a centrifuge
Centrifuge is used to hold
one phase stationary.
Countercurrent Chromatography
http://pubs.acs.org/subscribe/journals/tcaw/10/i07/html/07inst.html
under the
influence of
centrifugal force
CounterCurrent Chromatography
through the planetary motion of
coils (bobbins)

24.
CCS basicsK high
K = 1
K low
stationary phase
mobile phase
KD = conc. stationary/conc. mobile phase

25.
CCS basicsK high
K = 1
K low

26.
CCS basicsK high
K = 1
K low

27.
CCS basicsK high
K = 1
K low

28.
CCS basicsK high
K = 1
K low

29.
CCS 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
Q HD
FUE
C
V
r

30.
Countercurrent Separation Equations
VS = volume of stationary phase in the column
VM = volume of mobile phase in the column
VR = retention volume of analyte
K = partition coefficient
K =
Concentration of analyte in stationary phase
Concentration of analyte in mobile phase
K = (VR – VM)/VS
VC = total column volume = VM + VS

31.
Countercurrent Separation Equations
VC = total system volume
VS = volume of stationary phase in the column
VM = volume of mobile phase in the column
VR = retention volume of analyte
K = partition coefficient
Sf = stationary phase volume retention ratio
Sf = VS/VC = (VC – VM)/VC
K = (VR – VM)/VS = (VR – VM)/(Sf*VC)

32.
The separation factor (α) is the ratio of K values
for 2 consecutive peaks.
Countercurrent Separation Equations
𝛼 =
𝐾%
𝐾&
𝐾% > 𝐾&
The Resolution factor (RS) s another way to measure
the peak separation. W = peak width at the base
𝑅) =
%(+, - +.)
0,1 0.
http://www.shimadzu.com/an/hplc/support/lib/lctalk/resol-1.html

33.
The theoretical plates (N) is a way to measure
chromatographic efficiency. VR and W have the same
units.
𝑁 = 16(
𝑉6
𝑊
)%
Countercurrent Separation Equations
𝑅) =
&
8
𝑁
(+, - +.)
:;
<= :>
1
?,@ ?.
,
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.

34.
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.
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

35.
2
2
1 1
3
3
4
4
5 6
6
8 7
8
7
9
10
9 10
0.5 2 0.5 2
Normal
Phase
Reversed
Phase
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5 8
910
6
7
The CS Working Space
Non-standardized Standardized
Operation Parameters
1. CCC/CPC
2. CCC axes
3. rotational direction
4. synchronicity
5. winding left/right
6. hydrophilic UP/LP
7. hydrophilic VM/VS
8. winding & rotation
9. tail/head
10. K or 1/K
Friesen, J.B.; Pauli, G.F. (2009). "Binary concepts
and standardization in counter-current
separation technology". Journal of
Chromatography A 1216 (19): 4237–4244.
doi:10.1016/j.chroma.2009.01.048

36.
7
8
KD = 0.5
0 1 2
Lower (hydrophilic) Phase mobile
Head-to-Tail
Reversed phase elution
7
9
6
10
0 1 KD =2
Upper (lipophilic) Phase mobile
Tail-to-Head
Normal phase elution
7
9
6
10
Operation Parameters
FWD - IN
FWD – OUT
REV - IN
REV - OUT
Lower phase mobile
Upper phase mobile
HEMWat
Solvent
System
7
Friesen, J.B.; Pauli, G.F. (2009). "Binary concepts and standardization in counter-current separation technology". Journal of Chromatography A 1216 (19): 4237–4244.
doi:10.1016/j.chroma.2009.01.048

37.
Binary Choice Question
Answer for
example
Consequences
(6)
Aqueous
upper or lower?
lower
Free choice.
Any useable biphasic solvent
system is appropriate.
(8)
Do helical winding and
rotation
agree or oppose?
agree
Constrained choice.
Agreement gives optimal results.
(7)
Hydrophilic phase
mobile or stationary?
mobile
Free choice.
The analyte will elute from the
column with either choice.
(9)
Head-to-tail or
tail-to-head?
tail-to-head
Dependant on (3)
The direction of flow must
correspond to the choice of mobile
phase.
(7) Normal or reverse phase? normal phase
Dependant on (3)
The order of elution will correspond
on the choice of mobile phase.
(10)
Kshake-flask and KCCS; directly
or inversely proportional?
inversely
proportional
Dependant on (1) and (3)
The order of elution will correspond
on the choice of mobile phase.
Friesen, J.B.; Pauli, G.F. (2009). "Binary concepts and standardization in counter-current separation technology". Journal of Chromatography A 1216 (19): 4237–4244.
doi:10.1016/j.chroma.2009.01.048

38.
Parameter Type Parameter Experimental report
Essential Important Optional
Instrumental (All) Instrument make, model, and type
Column volumea
E
Sample loop volumea
I
Extra-column dead volume O
Back-pressure regulator setting O
Instrumental
(type-J specific)
Rotor radius (R) I
Range of spool radius (r) values I
β ratio (βr) I
Tubing inside diameter (bore) I
Tubing composition I
Head center/peripheral O
Length of tubing O
Number of turns per spool O
Direction of winding relative to
rotation
O
Instrumental
(CPC specific)
Rotor radius I
Channel number O
Channel volume O
Instrumental (Detector) Detector make, model, type E
Detector setting
(e.g. UV wavelength(s))
E
Flow cell details I
aAlthough column and sample loop volumes may be given by the
manufacturer, they can vary from instrument to instrument and should be
measured experimentally and reported as such
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 Instrumentational Parameters