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Centrifugal Partition Chromatography
What’s in the name: Countercurrent Chromatography
and Centrifugal Partition Chromatography?
 Generally,
the term Countercurrent Chromatography (CCC) covers all techniques,
in which components are separated as a result of partitioning a mixture of
components between two phases of a bi-phasic solvent system. Centrifugal
Partition Chromatography (CPC) is a sub-type of CCC, in
which the separation takes place in an axially rotating chamber that
contains hundreds of interconnected small mixing compartments. The most
recent addition to the family of the CPC instruments is
Kromaton®,
which distinguishes itself from the predeceasing CPC instruments by
achieving the flow rates several times higher and thus is being justly
called the FCPC®
(F stands for fast). For a 200 ml rotor, a typical flow rate is between 10
and 15 ml/min, for 1000 ml rotor between 30 and 100 ml/min. The high flow
rates allow faster completion of a run, which in-turn, leads to
substantial savings of time and costs.
CPC – How does
it work?
A
bi-phasic solvent system suitable for fractionation of a sample is
prepared based on literature data or own experiments. After selecting one
of the phases as the stationary, it is pumped into the FCPC® rotor, while
it is revolving at 200 rpm. The rotor contains 1,200 separate micro
compartments that are connected in series. The stationary phase is kept
immobilized due to a special design of the channels combined with the
effect of the centrifugal force. The design allows, however, for the
mobile phase to pass from one mixing chamber to another. An injected
sample passes through these compartments and in each of them, like in a
separatory funnel, the components of the sample partition between the two
phases. The rotational speed is increased to 700 rpm or higher for the
partitioning mode when a mobile phase is pumped into the rotor. When
equilibrium is established between the two phases, a sample is injected
and the solutes begin partitioning between the phases, much as they do in
the separatory funnel. Because the components partition between the two
phases differently, the mobile phase will carry out faster the components
with the smallest partition coefficients (p= [c] stationary
phase/[c] mobile phase). Thus, the component to be eluted first is the one
that partitions best into the mobile phase. On exit from the rotor, the
eluting mobile phase is directed to an appropriate detector and/or
fraction collector.
Among the most
notable advantages of the CCC separation methods are:
-
Absence of the irreversible
adsorption.
-
Recovery of all components of a
mixture intact. This feature is priceless when it comes to the
bioactivity-directed isolation of unknown compounds as it prevents an
unexpected loss of activity from fractionation.
-
Efficient purification of an
easily hydrolizing natural compound azadirachtin is a good illustration
of this gentle approach.
-
High load acceptance; 1L-rotor can
accept a sample of up to 30 grams and typical fractionation will consume
3 to 5 liters of a solvent system.
-
Selectivity that is unique, and
often very different from the solid phases such as silica gel or
ODS. It can be optimized for a given purpose by composing a solvent
system appropriately. In complex cases requiring multi-step purification
it can greatly complement other chromatographic techniques and as a rule
should be used as the first step purification. CPC can widely separate
the compounds that have the same retention times on the solid phase.
Good examples of the highly similar co-occurring in nature compounds
that co-elute on silica gel, but can be purified by CPC, are the pairs
Rotenone – Deguelin or Ruscogenin – Neoruscogenin.
-
Applicable to a wide range of
polarity of chemical compounds. From unsaturated hydrocarbons or lipids
on one end of polarity to the highly hydroxylated compounds such as
saponins, tannins, proanthocyanidins, or even quaternary alkaloids on
the opposite end of polarity.
-
Applicable to the water-sensitive
compounds. Several non-water containing solvent systems have been
described in literature (see: General References).
CPC for
natural products:
Natural product
extracts often present the greatest challenges in purification. Thus, it
is not surprising that the CCC methods have been embraced above all by the
natural products scientists. The crude extracts can be injected without
any prior cleaning.
Interestingly,
complete multi-step purifications of minor components can be completed
entirely by a series of consecutive CPC runs, exploiting different
selectivity of purposely composed solvent systems (e. g. isolation of two
potent immunosuppressants triptolide and tripdiolide from an extract of
Tripterygium wilfordii (J.A. Glinski
et al. "Bioassay-guided isolation of Triptolide from Tripterygium
wilfordii and its biological
properties. Thirty fourth Meeting of the American Society of
Pharmacognosy, July 18-22, 1993, San Diego CA).
General
References:
Countercurrent
Chromatography
Ed. Alain Berthod
Elsevier Health
Sciences, 2002
Countercurrent
Chromatography (Chromatographic Science, V. 82)
J.-M. Menet and D.
Thiebaut
Marcel Dekker,
1999
Countercurrent
Chromatography: Theory and Practice
Eds. N. B. Mandava
and Y. Ito
Marcel Dekker,
1988
High speed
countercurrent chromatography.
Eds. Yoshiro Ito
and Walter D. Conway
John Wiley & Sons,
Inc. New York, 1996
High-speed
Countercurrent Chromatography (Chemical Analysis, V.132)
Eds. E. Conway and
Y. Ito
Wiley-Interscience,
1995
Centrifugal
Partition Chromatography
Ed. A. Foucault
Marcel Dekker,
1994
Journal of Liquid
Chromatography, Vol. 13 (18) 1990
Ed. J. Cazes
Special Issue on
Centrifugal Partition Chromatography
Countercurrent
chromatography: apparatus, theory and applications.
Ed. Walter D.
Conway
VCH Publishers,
Inc. New York, 1990
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