The 9th International CCC Event will be held August 1-3, 2016 at Dominican University in River Forest, Illinois. The event will include conferences on August 1-3 and workshops on July 30-31. Brent Friesen, a Chemistry Professor at Dominican University, is the contact for the event. Countercurrent separation is a type of support-free liquid chromatography that has various instrumentation types including countercurrent chromatography, centrifugal partition chromatography, and droplet countercurrent chromatography. It provides advantages such as minimal sample preparation, high mass resolution, no sample loss, reproducibility, flexibility, and mild separation conditions for sensitive molecules.
Introduction to countercurrent chromatography: instruments
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
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
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