a brief introduction to countercurrent chromatography with its principle. working and modes of operation along with little bit of history, the types of CCC and its applications

1. Countercurrent chromatography
Compiled by-
Kaberi Nath
Roll no-17PBT206

2. Introduction
 Form of liquid–liquid chromatography that uses a liquid stationary phase that is held in
place by centrifugal force, and is used to separate, identify, and quantify the chemical
components of a mixture.
 It encompasses a collection of related liquid chromatography techniques that employ
two immiscible liquid phases without a solid support.
 The two liquid phases come in contact with each other as at least one phase is pumped
through a column, a hollow tube or a series of chambers connected with channels, which
contains both phases.
 The resulting dynamic mixing and settling action allows the components to be
separated by their respective solubilities in the two phases.
 A wide variety of two-phase solvent systems consisting of at least two immiscible liquids
may be employed to provide the proper selectivity for the desired separation.
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3. Components
 The components of a CCC system are similar to most liquid chromatography
configurations, such as high-performance liquid chromatography.
 One or more pumps deliver the phases to the column which is the CCC instrument
itself.
 Samples are introduced into the column through a sample loop filled with an
automated or manual syringe.
 The outflow is monitored with various detectors such as ultraviolet–visible
spectroscopy or mass spectrometry.
 The operation of the pumps, CCC instrument, sample injection, and detection may
be controlled manually or with a microprocessor.
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4. History
 The predecessor of modern counter-current chromatography theory and
practice was counter-current distribution (CCD).
 The theory of CCD was described in the 1930s by Randall and Longtin.
 Archer Martin and Richard Laurence Millington Synge developed the
methodology further during the 1940s.
 Finally, Lyman C. Craig introduced the Craig counter-current distribution
apparatus in 1944 which made CCD practical for laboratory work.
 CCD was used to separate a wide variety of useful compounds for several
decades.
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5. Support-free liquid chromatography
 In counter-current chromatography centrifugal or gravitational forces immobilize the stationary
liquid layer.
 By eliminating solid supports, permanent adsorption of the analyte onto the column is avoided,
and a high recovery of the analyte can be achieved.
 Solvent costs are also generally lower than for high-performance liquid chromatography (HPLC).
 In comparison to column chromatography, flows and total solvent usage can be reduced by half
and even up to a tenth.
 the cost of purchasing and disposing of stationary phase media is eliminated.
 experiments conducted in the laboratory can be scaled to industrial volumes.
 countercurrent chromatography operators can inject large volumes relative to column volume.
most modern commercial CCC can inject 5 to 40 g per litre capacity.
 When gas chromatography or HPLC is carried out with large volumes, resolution is lost due to
issues with surface-to-volume ratios and flow dynamics; this is avoided when both phases are
liquid.
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6. Principle
 The CCC separation process occurs in three stages: mixing, settling, and separation of the two phases
(although they often occur continuously).
 The analyte will distribute between the phases according to its partition coefficient (aka distribution
coefficient, distribution constant, or partition ratio) and is represented by P, K, D, Kc, or KD.
 The partition coefficient for an analyte in a particular biphasic solvent system is independent of the
volume of the instrument, flow rate, stationary phase retention volume ratio and the g-force required to
immobilize the stationary phase.
 The degree of stationary phase retention is a crucial parameter. factors that influence stationary phase
retention are flow rate, solvent composition of the biphasic solvent system, and the g-force.
 The stationary phase retention is represented by the stationary phase volume retention ratio (Sf) which is
the volume of the stationary phase divided by the total volume of the instrument.
 The settling time is a property of the solvent system and the sample matrix, both of which greatly
influence stationary phase retention.
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8. Working
 The counter-current separation starts with choosing an appropriate biphasic solvent
system for the desired separation.
 A wide array of biphasic solvent mixtures are available to the CCC practitioner including
the combination n-hexane (or heptane), ethyl acetate, methanol and water in different
proportions.
 Typically, it is desirable to choose a solvent system where the target compound(s) have
a partition coefficient between 0.25 and 8.
 After the biphasic solvent system has been chosen a batch of is formulated and
equilibrated in a separatory funnel (pre-equilibration of the solvent system).
 The two phases are separated. Then the column is filled with stationary phase with a
pump.
 Next, the column is set an equilibration conditions, such as the desired rotation speed,
and the mobile phase is pumped through the column.
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9.  The mobile phase displaces portion of the stationary phase until column equilibration is
achieved and the mobile phase elutes from the column.
 The sample may be introduced into the column at any time during the column
equilibration step or after equilibration has been accomplished.
 After the volume of eluant surpasses the volume of the mobile phase in the column, the
sample components will begin to elute.
 Compounds with a partition coefficient of unity will elute when one column volume of
mobile phase has passed through the column since the time of injection.
 The compound can then be introduced to another stationary phase to help increase the
resolution of results.
 The flow is stopped after the target compound(s) are eluted or the column is extruded
by pumping the stationary phase through the column.
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11. Droplet CCC
 Droplet countercurrent chromatography (DCCC) was introduced in 1970 by Tanimura,
Pisano, Ito, and Bowman
 DCCC uses only gravity to move the mobile phase through the stationary phase which is
held in long vertical tubes connected in series.
 In the descending mode, droplets of the denser mobile phase and sample are allowed
to fall through the columns of the lighter stationary phase using only gravity.
 If a less-dense mobile phase is used it will rise through the stationary phase; this is
called ascending mode.
 The eluent from one column is transferred to another; the more columns that are used,
the more theoretical plates can be achieved.
 DCCC enjoyed some success with natural product separations but was largely eclipsed
by the rapid development of high-speed counter-current chromatography.
 The main limitation of DCCC is that flow rates are low, and poor mixing is achieved for
most binary solvent systems.
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12. Hydrodynamic CCC
 The modern era of CCC began with the development of the planetary centrifuge by
Dr. Yoichiro
 It which was first introduced in 1966 as a closed helical tube which was rotated on a
"planetary" axis as is turned on a "sun" axis.
 A flow-through model was subsequently developed and the new technique was
called counter-current chromatography in 1970.
 The technique was further developed by employing test mixtures of DNP amino
acids in a chloroform : glacial acetic acid:0.1 M aqueous hydrochloric acid (2:2:1 v/v)
solvent system.
 Much development was needed to engineer the instrument so that required
planetary motion could be sustained while the phases were being pumped through
the coil(s).
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13. Hydrostatic CCC
 Hydrostatic CCC or centrifugal partition chromatography (CPC) was invented in the
1980s by the Japanese company Sanki Engineering Ltd, whose president was Kanichi
Nunogaki.
 CPC has been extensively developed in France starting from the late 1990s. In France,
they initially optimized the stacked disc concept initiated by Sanki.
 More recently, in France and UK, non-stacked disc CPC configurations have been
developed with PTFE, stainless steel or titanium rotors.
 These have been designed to overcome possible leakages between the stacked discs of
the original concept, and to allow steam cleaning for Good manufacturing practice.
 The volumes ranging from a 100 ml to 12 liters are available in different rotor materials.
 The 25-liter rotor CPC has a titanium rotor.
 This technique is sometimes sold under the name "fast" CPC or "high-performance"
CPC.
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14. Modes of operation
Normal-phase
 achieved by pumping the non-aqueous or phase of a biphasic solvent system through the column
as the mobile phase, with a more polar stationary phase being retained in the column.
Reversed-phase
 In reversed-phase (e.g. aqueous mobile phase) elution, the aqueous phase is used as the mobile
phase with a less polar stationary phase.
 In counter-current chromatography the same solvent system may be used in either normal or
reversed phase mode simply by switching the direction of mobile phase flow through the column.
Dual-mode
 the mobile and stationary phases are reversed part way through the separation experiment. This
requires changing the phase being pumped through the column as well as the direction of flow.
 Dual-mode operation is likely to elute the entire sample from the column but the order of elution
is disrupted by switching the phase and direction of flow.
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15. Dual-flow
 Dual-flow, aka dual countercurrent chromatography occurs when both phases are flowing in
opposite directions inside the column.
 Instruments are available for dual-flow operation for both Hydrodynamic and hydrostatic CCC.
 The countercurrent chromatography instrument must be modified so that both ends of the
have both inlet and outlet capabilities.
Gradient
 The use of a solvent gradient is very well developed in column chromatography but is less
in CCC.
 A solvent gradient is produced by increasing (or decreasing) the polarity of the mobile phase
during the separation to achieve optimal resolution across a wider range of polarities.
 For example, a methanol-water mobile phase gradient may be employed using heptane as the
stationary phase.
 This is not possible with all biphasic solvent systems, due to excessive loss of stationary phase
created by disruption the equilibrium conditions within the column.
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16. Elution-extrusion
 In elution-extrusion mode (EECCC), The mobile phase is extruded after a certain point by
switching the phase being pumped into the system whilst maintaining rotation.
 For example, if the separation has been initiated with the aqueous phase as the mobile phase
a certain point the organic phase is pumped through the column which effectively pushes out
both phases that are present in the column at the time of switching.
 The complete sample is eluted in the order of polarity without loss of resolution by diffusion.
Recycling and Sequential
 Recycling chromatography is mode practiced in both HPLC and CCC.
 the target compounds are reintroduced into the column after they elute.
 Each pass through the column increases the number of theoretical plates the compounds
experience and enhances chromatographic resolution.
 Direct recycling must be done with an isocratic solvent system.
 With this mode, the eluant can be selectively re-chromatographed on the same or a different
column in order to facilitate the separation.
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17. Ion-exchange and pH-Zone-refining
 An ion-exchange mode has been created by modifying both of the phases after pre-
 Generally, an ionic displacer (or eluter) is added to mobile phase and an ionic retainer is added
the stationary phase.
 For example, the aqueous mobile phase may contain NaI as a displacer and the organic
phase may be modified with the quaternary ammonium salt called Aliquat 336 as a retainer.
 The mode known as pH-zone-refining is a type of ion-exchange mode that utilizes acids and/or
bases as solvent modifiers.
 Typically, the analytes are eluted in an order determined by their pKa values.
 Ion-exchange modes such as pH-zone-refining have tremendous potential because high sample
loads can be achieved without sacrificing separation power. It works best with ionizable
compounds such as nitrogen containing alkaloids or carboxylic acid containing fatty acids.
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18. Applications
 Countercurrent chromatography and related liquid-liquid separation techniques
have been used on both industrial and laboratory scale to purify a wide variety of
chemical substances.
 Separation realizations include proteins, DNA, antibiotics, vitamins, natural
products, pharmaceuticals, metal ions, pesticides, enantiomers, polyaromatic
hydrocarbons from environmental samples, active enzymes, and carbon
nanotubes.
 Countercurrent chromatography is known for its high dynamic range of scalability:
milligram to kilogram quantities purified chemical components may be obtained
with this technique.
 It also has the advantage of accommodating chemically complex samples with
undissolved particulates.
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19. References
 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). doi:10.1351/PAC-REP-08-06-05. ISSN 1365-3075
 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.
 High-speed countercurrent chromatography. Chemical analysis. Yoichiro Ito, Walter D. Conway (eds.). New York: J. Wiley. 1996. ISBN 978-0-471-63749-3.
 Ito, Yuko; Goto, Tomomi; Yamada, Sadaji; Matsumoto, Hiroshi; Oka, Hisao; Takahashi, Nobuyuki; Nakazawa, Hiroyuki; Nagase, Hisamitsu; Ito, Yoichiro (2006).
"Application of dual counter-current chromatography for rapid sample preparation of N-methylcarbamate pesticides in vegetable oil and citrus fruit". Journal of
Chromatography A. 1108 (1): 20–25.
 Tanimura, Takenori; Pisano, John J.; Ito, Yoichiro; Bowman, Robert L. (1970). "Droplet Countercurrent Chromatography". Science. 169: 54–56.
Bibcode:1970Sci...169...54T. doi:10.1126/science.169.3940.54.
 Foucault, Alain P. (1994). Centrifugal Partition Chromatography. Chromatographic Science Series, Vol. 68. CRC Press. ISBN 978-0-8247-9257-2.
 Marchal, Luc; Legrand, Jack; Foucault, Alain (2003). "Centrifugal partition chromatography: A survey of its history, and our recent advances in the field". The
Chemical Record. 3 (3): 133–143. doi:10.1002/tcr.10057.
 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.
 Randall, Merle; Longtin, Bruce (1938). "SEPARATION PROCESSES General Method of Analysis". Industrial & Engineering Chemistry. 30 (9): 1063–1067.
doi:10.1021/ie50345a028. Retrieved 2016-02-22.
 Lyman C. Craig (1944). "Identification of Small Amounts of Organic Compounds by Distribution Studies: II. Separation by Counter-current Distribution". Journal of
Biological Chemistry. 155: 535–546.
 Kimura, Yukio; Kitamura, Hisami; Hayashi, Kyozo (1982). "A method for separating commercial colistin complex into new components: colistins pro-A, pro-B and
pro-C". The Journal of Antibiotics. 35 (11): 1513–1520. doi:10.7164/antibiotics.35.1513. Retrieved 2016-02-22.
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