Counter current chromatography

Counter current chromatography (CCC) is a liquid chromatography technique that
uses two immiscible liquid phases and no solid support.
2. One liquid acts as the stationary phase and the other as the mobile phase.
3. In Dual Flow CCC/CPC both liquid phases are flowing, as would be common in counter
current process extractors.
4. The liquid stationary phase(s) is held in place by gravity or by centrifugal force. The
gravity method is called droplet counter current chromatography (DCCC).
5. There are two modes of centrifugal force CCC: hydrostatic and hydrodynamic. In the
hydrostatic method.
6. The column is spun about

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Counter current chromatography
Mr. Sagar Kishor Savale
[Department of Pharmacy (Pharmaceutics)]
2015-016
[email protected]

1. Introduction
1. Counter current chromatography (CCC) is a liquid chromatography technique that
uses two immiscible liquid phases and no solid support.
2. One liquid acts as the stationary phase and the other as the mobile phase.
3. In Dual Flow CCC/CPC both liquid phases are flowing, as would be common in counter
current process extractors.
4. The liquid stationary phase(s) is held in place by gravity or by centrifugal force. The
gravity method is called droplet counter current chromatography (DCCC).
5. There are two modes of centrifugal force CCC: hydrostatic and hydrodynamic. In the
hydrostatic method.
6. The column is spun about a central axis. These devices are marketed under the
commercial name centrifugal partition chromatography CPC).
7. Dynamic mode is often called high-speed CCC (HSCCC) and relies on the Archimedes’
screw force in a helical coil to produce the separation.
2. History
1. The inventor Yoichiro Ito (1978), named it after the countercurrent partition method of
Craig.
2. Countercurrent chromatography (CCC) is a liquid chromatography (LC) technique.

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3. Principle
1. Countercurrent chromatography or partition chromatography is liquid-liquid
chromatographic technique.
2. Separate components of a mixture based on their differing affinities for mobile and
stationary phases of a column.
3. Here stationary phase and mobile phase are both liquid. Partition of solute in two phases
is based on differences in capacity factor, k, and distribution coefficient, Kd. of the
analyte using stationary liquid and mobile liquid phase.
4. Stages of Counter Current Chromatography
1. Mixing
2. Settling
3. Separation
5. Importance
1. Distribution constant D.
2. Stationary phase retention volume.
3. Elution time.
4. Resolution.
5. Effect of column length.

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6. Instrumentation

7. Modes of Operation
1. Head to tail
2. Tail to head
3. Dual-Mode
4. Gradient Mode
5. pH Zone Refining
8. Types of Counter current Chromatography
1. Hydrostatic CCC columns
2. Hydrodynamic CCC columns

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8.1 Hydrostatic column design

8.2 Hydrodynamic column design

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Table 1 Comparison of the properties of hydrodynamic and hydrostatic columns.
Column Hydrodynamic Hydrostatic
Liquid retained in Coiled Teflon tubes channels
Commercial name & acronym Coil planet centrifuge, high-sped
CCC
Centrifugal partition
chromatography

Centrifugal field Variable, two axes of rotation Constant one axis of rotation
Stationary phase retention Variable Good
Efficiency Up to 4 plates per tube run Up to 1 plate per channel
Pressure Low, 0.1-10 kg/cm2 Medium, 2-70 kg/cm2
Maintenance Connecting tubes to change every
~100 h
Rotating tubes to lubricate every
~100 h
other Possible noisy gear assembly,
quieter centrifuge with belt drive
Quiet centrifuge

9. Types of Counter Current Chromatography
1. Droplet counter current chromatography.
2. Elution Extrusion Counter Current Chromatography
3. Centrifugal Partition Chromatography
4. High Speed Counter Current Chromatography.
1. Droplet counter current chromatography
1. It uses only gravity to move the mobile phase through the stationary phase.

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2. In descending mode, droplets of the denser mobile phase and sample are allowed to fall
through a column of the lighter stationary phase using only gravity.
3. If a less dense mobile phase is used it will rise through the stationary phase, this is
called ascending mode.
4. The eluent from one column is transferred to another; the more columns that are used,
the more theoretical plates can be achieved.

2. Elution Extrusion Counter Current Chromatography
1. When run reaches a certain point, mobile phase is stopped & stationary phase is pumped
in to extrude the column content.
2. Elution extrusion makes use of the fact that compounds may be fully separated inside
the column before eluting from it. Due to the fact we are using a liquid SP, we are able
to recover the separated compounds without completing the full elution cycle.

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3. In elution extrusion, the separation is started in the same manner as in single-mode
CCC.
4. However, when the run reaches a certain point (e.g. D=1.0), MP will be stopped and
stationary phase pumped in to extrude the column contents (i.e. the phase initially used
as SP will be introduced in the instrument as a new MP).
3. Centrifugal Partition Chromatography
1. Invented by Sanki engineering ltd, Japan in 18
th
cent.
2. CPC use CENTRIFUGAL FORCE to speed separation and achieves higher
flow rates than DCCC.
3. The centrifugal partition chromatograph is constituted with a unique rotor
(column). This rotor rotates on its central axis.
4. With less vibrations and noise, the CPC offers a wider rotation speed range
(from 500 to 2000 rpm) than HSCCC.
5. That allows a better decantation and retention for unstable biphasic system (e.g.,
aqueous aqueous systems or Butanol/water systems). Basics of CPC: The CPC
rotor is constituted by the superposition of disks engraved with small cells
connected by head / tail ducts.
6. These are the cells, where actual separation takes place.
7. Rotor is filled with the stationary phase, which stays inside the rotor, while
mobile phase is pumped through.
4. High-Speed countercurrent chromatography

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1. High-Speed Countercurrent Chromatography (HSCCC) instruments are a modern
and popular type of Countercurrent Chromatography (CCC) apparatus. These
machines consist of:
2. Helically coiled inert tubing (Usually Teflon or sometimes stainless steel) that rotates
on its own axis.
3. A gear assembly arranged such that the helical coils revolve around a central axis to
achieve planetary motion.
10. Advantages
1. It is simple, rapid, and reproducible.
2. High sensitivity.
3. High performance.
4. Rapid process and hence time saving.
5. It is having a high resolution and separation capacity.
6. Accuracy and Precision.
7. Stationary phase was chemically innert.
8. Wide varities of stationary phase.
9. Mobile phase was chemically innert.
10. Less requirement of mobile phase in developing chamber.
11. Early recovery of separated component.
12. Easy visualization of separated components.
13. It is having Good reproducibility and repeatability.
14. It is analytical technique is important for validation of product, quality control studies
of product.
15. It is important for qualitative and quantitative analysis.

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11. Applications
1. Analysis of plant and different natural products.
2. Food analysis.
3. Environment analysis.
4. Inorganic ions
5. Lanthanides and actinides
6. Alkali and alkaline-earth elements
7. Platinum group metals
8. Various other elements
9. Purification of salt solutions by CCC
10. Recovery of PAHs from liquid sewage sludge and soil
12. Reference
1. Conway WD (1990) Countercurrent Chromatography, Apparatus Theory and
Applications. New York: VCH
2. Conway WD and Petroski RJ (eds) (1995) Modern Countercurrent Chromatography.
ACS Symposium Series 593. Washington, DC: American Chemical Society.
3. Conway WD, Bachert EL, Sarlo AM and Chan CW (1998) Comparison of
countercurrent chromatography with Sash chromatography. Journal of Liquid
Chromatography and Related Technologies 21: 53}63.

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4. Counter Current Chromatography: A Superior and An Advanced Analytical Technique
by Naman B. Doshi, Avani H. Sheth, Dr Dhrubo Jyoti Sen and Dr J B Dave Pharma
Times - Vol 42 - No. 01 - January 2010.
5. www.dynamicextractions.com
6. Berthod, Alain; Maryutina, Tatyana; Spivakov, Boris; Shpigun, Oleg; Sutherland, Ian
A. (2009). “Countercurrent chromatography in analytical chemistry (IUPAC Technical
Report)". Pure and Applied Chemistry 81 (2): 355–387. doi:10.1351/PAC-REP-08-06-
05. ISSN 0033- 4545.
7. Ito, Y.; Bowman, RL (1970). “Countercurrent Chromatography: Liquid-Liquid
Partition Chromatography without Solid Support”. Science 167 (3916): 281–283.
Bibcode:1970Sci...167..281I. doi:10.1126/science.167.3916.281. PMID 5409709.
8. Foucault, Alain P. (1994). Centrifugal Partition Chromatography. Chromatographic
Science Series, Vol. 68.CRC Press. ISBN 978-0824792572.
9. Ito, Yoichiro (2005). “Golden rules and pitfalls in selecting optimum conditions for
high-speed counter-current chromatography”. Journal of Chromatography A 1065 (2):
145–168. doi:10.1016/j.chroma.2004.12.044. ISSN 0021-9673.
10. Ian A. Sutherland (2007). “Recent progress on the industrial scale-up of counter-current
chromatography” Journal of Chromatography A 1151: 6–13.