Plants

Chapter 9
Separation of Natural Products by Countercurrent

Chromatography
James B. McAlpine, J. Brent Friesen, and Guido F. Pauli
Abstract
Countercurrent Chromatography (CCC) provides the natural product chemist with a high-resolution
separatory method, which is uniquely applicable to sensitive (unstable) compounds and which allows
virtually quantitative recovery of the load sample. Different instruments use different means of retaining a
stationary liquid phase. The solvent system (SS) can be chosen to optimize the separatory power and the
number of systems available is limitless. Several examples are provided to illustrate the power of the method
and to guide the chemist in choice of an appropriate SS.
Key words: High Speed Countercurrent Chromatography (HSCCC), Centrifugal partition chromatography, Itos coil planet centrifuge, pH zone refining, GUESS method, Two-phase solvent systems
1. Introduction
Modern countercurrent chromatography (CCC) has arisen only
over the last 30 or so years and offers the natural product chemist
a further separation tool with many unique advantages. It is inherently
the mildest form of chromatography with no solid support and
hence no chance of loss of substrate by binding to the stationary
phase. The only media encountered by the sample are solvent and,
typically, Teflon tubing. The former is common to all forms of
chromatography and the latter to most. It is true that many of the
solvents systems (SSs) have more components than most other
forms of chromatography, but these can be chosen from the most
nonreactive and innocuous solvents. Hence, the chromatographer
is virtually assured of near 100% recovery of sample from a chromatography. The number of two-phase systems, which can be
employed, is limited only by the imagination of the chromatographer,
Satyajit D. Sarker and Lutfun Nahar (eds.), Natural Products Isolation, Methods in Molecular Biology, vol. 864,
DOI 10.1007/978-1-61779-624-1_9, Springer Science+Business Media, LLC 2012
221
222
J.B. McAlpine et al.
and the systems can be explored by any of several simple tests, prior
to a preparative separation, to assure success. Two similar compounds of almost identical polarity can have surprisingly different
partition coefficients in a specific two-phase system resulting in
base-line separation by CCC.
CCC had its beginning in the 1950s with the Craig machine
(1), a mechanical system of sequential separating cells in which one
phase of a two-phase SS could be equilibrated with the other phase
in successive cells thereby carrying a solute along according to its
partition coefficient between the two phases. Solutes would be
washed from the train in the order that their partition coefficients
favored the mobile phase. These instruments were cumbersome,
delicate, and required a major ventilation system as they invariably
leaked organic solvents to some extent. A typical system would
involve 200400 cells and a separation using such an instrument
would take in the order of a week to accomplish. They were used
because they could achieve separations, which could not be otherwise effected. They were both displaced and replaced. Displaced by
the high pressure liquid chromatography (HPLC) and the large
number of possible solid supports available for this methodology,
and replaced by droplet countercurrent (DCCC) and rotary locular
(RLCCC) instruments, which could be used to effect that same
separation method in a fraction of the laboratory space. DCCC
and RLCCC instruments are still available (Eyela). The former
composed of vertical tubes of a diameter that will allow droplets of
one phase of a two-phase SS to rise (or fall) through the other
phase. These mixing tubes are then connected top to bottom, with
fine tubing such that the droplets would completely fill the tubing
and exclude the stationary phase from these interconnecting tubes.
Thus, the mobile phase is added slowly and allowed to percolate
through the mixing chambers under the force of gravity, to achieve
a distribution of solute according to its partition coefficient. DCCC
systems, while sharing the high separatory power of the Craig
machine and the high overall recovery of load sample, still suffer
from the problem of being slow, and this is further complicated by
the need to maintain constant temperature during the course of a
chromatography.
However, a very recent report by Dini demonstrates that this
can still be a highly effective method of separation (2). The instrument used was a Bchi DCCC equipped with 300 2.2 mm i.d.
tubes each with 2.0 mL volume (according to anecdotal evidence,
only around 50 machines were built, and very few are probably still
operable; another one is located in the chapters authors laboratory). Using a stationary phase of n-butanol/acetone/water (60:12:28)
and a mobile phase of n-butanol/acetone/water (14:12:74), three
new dihydrokaempferol glycosides (Fig. 1) were isolated and characterized from a single run (2).
Separation of Natural Products by Countercurrent Chromatography
OR
HO
O
223
R=H
R = L-xylopyranoside
OCH3
OH
O
OCH3
O
HO
OH
OH
O
OH
HO
Kaempferol glycosides from Pouteria obovata
OH
OH
HO
O
OH
OH
O
O
OAc
R"
OH
HO
HO
Gamabufotalin R = OH, R' = R" = H

Telocinobufagin R = R"= H, R' = OH
Bufotalin R = R' = H, R" = OAc
Bufalin R = R' = R" = H
O
HO
OH
R'
Cinobufatolin
OH
HO
H
Arenobufagin
Fig. 1. Structures of flavonoid glycosides from Pouteria obovata and bufadienolides isolated from Chan Su.
High-speed CCC (HSCCC) instruments became commercially

available around 1980 and have overcome all of the drawbacks of
these earlier instruments. They make CCC a useful means of
achieving delicate separations on the milligram to kilogram scale in
a few hours. Over the 7 years since the last edition of this book was
written, several new innovations in CCC methodology, have been
introduced and make CCC a highly efficient means of isolation/
purification of active principles from natural product extracts. Several
reviews covering various aspects of the technology have appeared.
Hostettmann and Marstons early review (3) emphasized phytochemicals and covered examples from both fast centrifugal partition chromatography, commonly referred to as FCPC, and the coil
planet centrifuge. Their later review (4) reflects the movement of
the community toward the latter instrument, and like the earlier
review is directed entirely at phytochemicals. Itos review (5) was
also concentrated on the J-type coil planet centrifuge, covers different
SSs and provides an extensive list of examples from rare earth elements to enzymes. Stichers review (6) of natural product isolation
224
covers phytochemicals very comprehensively, and Sutherland and

Fishers review (7) covers the role CCC plays in the modernization
of Chinese herbal medicine. A review by Pan and Lu (8) covers
extensively the literature on use in the last 10 years of several different Ito machines. The most recent review by Yoon et al. (9) is a
comprehensive coverage of the use of the centrifugal partition
systems over the last 15 years, predominantly for phytochemicals.
It is arranged by chemical classes and has tables of systems used for
each class. However as one small section is devoted to antibiotics it
does include some microbial metabolites. Perhaps the most comprehensive review of the methods and instrumentation appeared in
the Journal of Natural Products, although much of the material is
in the Supporting Information PDF file (10).
This chapter offers the reader a primer in the use of CCC techniques for separations and purifications in natural product isolations.
2. Materials
2.1. Current
Instruments
Although several minor variants are available, instruments are basically of two types.
(a) The Centrifugal Partition Chromatography instrument as sold
for many years by Sanki Instruments (Kyoto, Japan) and more
recently by Armen (Saint Ave, France) and Rousselet Rotatel
Kromaton (Annonay, France).
(b) The Coil Planet Centrifuge as designed by Yoichiro Ito.
Variants of the latter are currently sold by AECS (Bridgend,
UK), Conway Centri Chrom Inc (Williamsville, NY, USA), Cherry
Instruments (Chicago, USA), Dynamic Extractions Ltd. (Uxbridge,
UK), and Tauto Biotech (Shanghai, China). Originally, the acronym
CPC was used by Ito for his Coil Planet Centrifuge, which acts in
hydrodynamic mode. Only later CPC was used to designate the
hydrostatic Centrifugal Partition Chromatography.
The Centrifugal Partition instruments effectively replace gravity
as the driving force of a DCCC procedure with centrifugal force
and, thus, achieve a remarkable increase in the speed of the process.
This does necessitate the use of two rotating seals. The Teflon
tubing is replaced by small solid blocks or sheets of Tefzel, honeycombed with a channel system analogous to the tubing of a droplet
system. A distinct advantage of this system is the ability to use relatively viscous solvents such as n-butanol at r. t. Although n-butanolcontaining SSs can be used in the Coil Planet Centrifuge, they
usually result in poor retention of stationary phase at ambient
temperatures, and hence sub-optimal performance, unless other
components reduce the viscosity of the n-butanol-containing phase.
An obvious weak point in the design of the Centrifugal Partition
225
Chromatograph is the rotating seal, which must remain solvent

resistant and leak-proof to a wide variety of solvents under speeds
as high as 2,000 rpm. Experiments with crude extracts containing
small solid particles are particularly deleterious to these seals. The
authors limited experience with ceramic seals would suggest that
these are surprisingly robust.
The Coil Planet Centrifuge, commonly referred to as a J-Type
instrument, is just one of a large number of instruments which
have been the life work of Yoichiro Ito. His study of the movement
of one phase of a two-phase SS with respect to the other under a
variety of imposed vectors, and the use of this behavior as a separatory tool is without equal. The Coil Planet Centrifuge is available
both in horizontal and vertical configurations, and although the
forces imposed on the solvents are slightly different, the practical
effects are essentially equivalent and the two systems can be used
interchangeably to effect a separation. It consists of Teflon tubing
wound in a spiral around a central cylinder. When the coil is filled
with liquid and spun around its axis, an Archimedean screw force
is exerted on the liquid tending to drive it toward the center (head)
of the spiral or toward the outside (tail) depending on the direction of spin. This Archimedean screw force provides the counter to
the flow, and the means by which one phase is held stationary while
the other is pumped through it. Moreover, if the coil is spun in a
synchronous planetary motion such that the period of orbital
rotation is the same as that for spin around the axis of the coil, it is
possible to thread the feed and exit lines through the center spindle
of the coil and out the center spindle of the orbit. These two lines
do not entwine as the instrument spins and hence the need for a
rotating seal is removed. In practice, the orbital axis is the drive axis
and the coil spin is driven from this axis by two identical cogs.
Given that the usual operating speed is from 800 to 2,000 rpm and
that a single chromatography can takes up to 3 h and therefore
involves in excess of 100,000 rotations, it is not surprising that
the inlet and outlet do become twisted with constant use. It is
necessary to inspect and occasionally untwist them. For an instrument in steady use this needs only to be done about once a month
or even less frequently and should never be done while the instrument is in motion.
The two-phase system undergoes some interesting dynamics
during operation as explained theoretically by Ito (11) and demonstrated by strobe light photography by Conway and Ito (12).
Within each orbital rotation, the two phases undergo a mixing
and a separation step. This is reasonably postulated to increase the
partition efficiency, and hence the separatory resolution of the method.
Very recently the Brunel/Dynamic Extractions group have come
up with non-synchronous planet coil centrifuge design which also
does not require rotating seals and which allows for differential
control of the mixing and settling stages of the chromatography (13).
226
When commercial instruments of this design become readily available,

they should take separation science to a new quantum level of
efficiency.
Typical semi-preparative instruments of both types hold
between 100 and 300 mL in a column, however analytical coils are
available for the Coil Planet Centrifuge with a capacity as low as
20 mL, and are common, as are larger coils of capacity 1 L or
more. A prototype, 18 L machine has recently been built by
Dynamic Extractions and shown to produce resolution comparable
to that of analytical machines (14, 15). The early instruments,
which had a single coil, had to be counterbalanced, and the counterbalance had to be tailored to the SS. Newer instruments have as
many as four identical coils connected in series and symmetrically
placed around the central axis. This provides an internal counterbalance, however the system is only in balance to the extent that
each coil is maintaining the same amount of stationary and mobile
phases. The difficulty with increasing the scale lies in the large centrifugal forces generated and the need to keep such a system in
balance as the SS changes in composition while filling the columns
or if any significant bleeding of stationary phase should occur during a chromatography.
2.2. Current Vendors
CCC has not caught the attention of the large instrument makers, and
their manufacture tends to be a cottage industry. Currently, instruments are available from the following suppliers/manufacturers.
(a) AECS, PO Box 80 Bridgend, South Wales, United Kingdom
CF31 4XZ. Tel: 44-783-876-1524, Fax: 44-1656-789-282,
E-mail: aecs@gmx.com.
(b) Armen Instrument, Z.I de Kermelin, 16 Rue Ampre, F-56890
Saint Ave, France, Tel: 33 (0) 2 97 61 84 00, Fax: 33 (0) 2 97
61 85 00, E-mail: contact@armen-instrument.com.
(c) Cherry Instruments 3440 S. Dearborn St. Suite 242 Chicago,
IL 60616. Email: contact@cherryinstruments.com, Tel: 1-800852-0955 or 1-773-260-0204.
(d) Conway Centri Chrom Inc., 52 MacArthur Drive, Williamsville,
NY 14221. Tel: 1-716-635-088 Fax: 1-716-635-0886. E-mail:
wdconway@centrichrom.com.
(e) Dynamic Extractions Ltd Berkshire, UK, Tel: 44-1753 696979.
WWW: http://www.dynamicextractions.com.
(f) Eyela/Tokyo Rikakikai, c/o Dychrom, 190 Mountain ViewAlviso Road, Suite T, Sunnyvale, CA 94089 USA, Tel: 408745-7911, Fax: 408-745-1554, T. 1-800-439-2476, E-mail:
frank.tao@dychrom.com (DCCC, RLCCC).
(g) Rousselet Robatel Kromaton ZI, Marenton 45, Avenue Rhin
& Danube 07100, Annonay, France, Tel: 33(4)-75-69-22-11
Fax: 33(4) 75-67-69-80 E-mail: kromaton@kromaton.com.
227
(h) Shanghai Tauto Biotech Co., Ltd. Room A301 No. 326 Aidisheng
Road, Zhangjiang Hi-Tech Park, Shanghai, Peoples Republic
of China, 201203. Tel: 86-21-51320502. E-mail: export1@
tautobiotech.com.
3. Methods
3.1. Solvent Systems
and GUESS
Methodology
3.1.1. The Nature
of Solvent Systems
3.1.2. The Development

of GUESS Methodology
The most critical step in a CCC is the choice of the two phase SS
and these commonly consist of an aqueous and an organic phase,
although this is not necessarily the case. Non-aqueous two phase
systems have been used as have systems in which both phases are
aqueous (4, 1620). The chromatography can be run in a normal
phase mode, i.e., the polar (usually aqueous) phase is stationary or
in a reversed-phase mode with the aqueous phase mobile. This
interchange can be achieved by changing the direction of spin or
the direction of flow through the instrument. Dichloromethane
(DCM) and diethyl ether can both be used but the researcher
should be aware that a vapor lock will force the stationary phase
from the system, and abort the chromatography. Hence these
solvents need to be used only if the instrument comes with temperature control or if ambient temperature permits.
The utility of TLC in selection of an SS in the HEMWat and
ChMWat systems has been greatly increased by development of the
GUESS (Generally Useful Estimate of Solvent Systems) methodology (21, 22), which further exemplifies the analogy between CCC
and TLC (see Note 1 and Tables 13). The GUESS methodology
was developed by taking 21 commercially available natural products of widely varying polarity (The GUESS Mix) and demonstrating that their Rf values on a normal phase silica TLC developed
with the lipophilic phase of a potential SS correlated with their
partition coefficient in that SS. Hence the scientist runs a normal
phase silica gel TLCs of the analyte using only hexane and EtOAc
in the ratios they occur in some of the standard HEMWat systems,
say 4, 0 and +4 (i.e., 7:3, 1:1, and 3:7 respectively). The aim is to
find a ratio which results in the analyte having an Rf of ~0.5. The
corresponding HEMWat system will very probably partition the
analyte with a partition coefficient of between 0.25 and 4.0, and
hence will place the analyte in the sweet spot of a CCC run. The
usefulness of the GUESS mix has been expanded to include not
only solvent system prediction in HEMWat solvent systems but
also the systematic analysis and comparison of biphasic solvent systems commonly employed in CCC. The wide polarity range of the
GUESS mix components enables mapping of optimal polarity
ranges for HEMWat, EBuWat, terAcWat, and HterAcWat solvent
system families.
228
Table 1
Abbreviations of common constituents used for the
nomenclature of 2-phase CCC solvent systems (21)
Solvent
Abbreviation
Acetic acid
Acetone
At
Acetonitrile
Ac
n-Butanol
Bu
t-Butylmethyl ether
Ter
Chloroform
Ch
Cyclohexane
Cy (or Cyc)
Dichloromethane
D (or Di)
Diethylether
De
Ethanol
Et (or Eta)
Ethyl acetate
Hexane
H (or He)
Heptane
Hep
Methanol
M (or Me)
Toluene
Tol
Water
Wat
When two abbreviations are given, the second one can be used when it is
necessary to add consonance to the SS name
As the GUESS methodology has become more widely accepted,

subsets of the original GUESS mix have also been used. For
example, in Guzlekss study (23) comparing performance of
different instruments, it comprised only 12 compounds, four of which
were transparent in their detection system. The effectiveness of a
novel separation method known as intermittent countercurrent
extraction was demonstrated with a four component GUESS mix.
A conventional twin column CCC was employed to split a sample
into two groups of two compounds that were eluted from either
end of the instrument at regular intervals (24).
A four component sub-set of the GUESS mix was used by the
Sutherland group, both to show high loading capacity and easy
scale-up from a 912 mL to a 4,600 mL hydrodynamic CCC instrument, and the feasibility of scale up from a 120 mL Pharma-Tech
instrument to a 9 L Dynamic Extraction New Maxi (14, 15).
3.2. Solvent System
Selection
In the course of natural product isolation, the chemist is often

presented with mixtures containing very closely related biosynthetic
229
Table 2
The HEMWat family of two-phase solvent systems (SSs)
consisting of n-hexane (H), ethyl acetate (E), methanol (M), and
water (Wat) represents the most widely used SS in the literature
HEMWat#
Wat
+1
+2
+3
+4
+5
+6
+7
+8
10
10
The equivolumetric mixture, HEMWat 0, represents the portal SS of the family,

which can be varied systematically in polarity according to this table
Table 3
The ChMWat family of two-phase SSs All ChMWat systems
include 10 volumes of chloroform
ChMWat#
Wat
10
+1
+2
+3
+4
230
relatives, which may differ only by one or two methylenes, the

placement of an olefin, or the stereochemistry of a non-polar substituent. If the molecules have strongly polar groups common to
their structure, the difference in polarity associated with these structural differences can be insignificant, and render an adsorption type
chromatographic method useless. In this situation CCC is often the
only viable separation method, especially if the mixture has already
been the subject of multiple chromatographic steps. Although the
methods given above for choice of a SS may well work, this may be
the time to employ more sophisticated analytical techniques to
ensure success. TLC or analytical HPLC of each phase in the twophase system will work if the congeners are sufficiently separated in
such a system to assay them but if not, it is worth examining the two
phases by quantitative 1H-NMR or an HPLC-MS assay to ascertain
the partition coefficients of the congeners with a high level of confidence. Another alternative is to simply run an analytical CCC in
trial systems. The recently introduced 19 mL coil from Dynamic
Extractions (25) and the 20 mL Tauto instrument are ideal for this.
A cursory glance at the SSs used in Table 4 will reveal that
most of the two-phase systems are multi-component, and that
researchers have used their ingenuity in exploring a wide range of
components including almost any solvent that can easily be removed
from the analyte at the end of the chromatography. The initial
isolation of a natural product may be attempted with the only
knowledge of the active principle being its bioactivity. Under these
circumstances the aforementioned GUESS methodology is only
applicable if a TLC bioautography method (see Chapter 6) is available.
If not, the bioactivity itself must be the analysis for determining
the partition coefficient. Here a crude partition coefficient can be
determined by distributing the mixture in the two-phase system
and bio-assaying each phase. Partition coefficients based on bioactivity of crude mixtures have the inherent problem that several
congeners of a natural product extract may be bioactive, and the
determined value is a weighted mean based on potencies, quantities and partition coefficients of individual components. Although
this is theoretically disturbing, in practice it seldom seems to
present a problem as, one of two situations will prevail. Either the
congeners will have similar partition coefficients, then one will
choose a SS in which they fall in the area of maximum resolution
(the sweet spot), leading to separation from other components
in the mixture, and probably from one another. Alternatively,
the bioactive components will have widely different partition
coefficients, and the system will be useful in separating them from
one another, even if less effective in separating them from other
components. Our experience here is that 10 widely different
solvent systems can be rapidly tested and assayed. Not infrequently
this approach provides a real bonus, when one discovers two
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Table 4
Application of CCC in natural products isolation
Solvent system
Compounds
References
Light-PetEtOAcCCl4CHCl3MeOHH2O
1:1:4:4:6:2
IsooctaneEtOAcMeOHH2O 7:3:6:4
n-HexaneEtOAcMeOHH2O 70:30:15:6
Liensinine alkaloids (Fig. 2)
(43, 44)
Taxol (Fig. 4)
Tirandamycins A and B (Fig. 6)
(57)
(63)
Arizonins, concanamycins (Fig. 7)
(64, 65)
n-HexaneEtOAcMeOHH2O (0.01 N H2SO4)

5:6:5:6
Squalestatins (Fig. 6)
(66)
Arizonins (Fig. 7)
(64)
Auxins (Fig. 8)
(67)
n-HexaneEtOAcMeOHH2O (25 mM
phosphate buffer pH 6.9) 7:3:5:5
Australifungins (Fig. 9)
(68)
to 10:15:8:10 step-wise
n-HexaneEtOAcEtOHH2O 8:2:7:3
Phomopsolides (Fig. 8)
Bufadienolides (Fig. 1)
(69)
(26)
Tanshinones (Fig. 5)
(60)
n-HexaneEtOAcMeOHEtOHH2O
10:14:10:2:13
Taxol and cephalomannine (Fig. 4) (56)
n-HexaneCHCl3MeOHH2O 1:1:1:1
Trichoveroids (Fig. 10)
(70)
n-HexaneDCMMeOHH2O 5:1:1:1
Bu 2313 B (A tetramic acid),

5-N-acetylardeemin (Fig. 11)
(63, 71)
n-HexaneDCMMeOHH2O 10:40:17:8
n-Hexanen-BuOHH2O (0.05%TFA) 43:7:50
n-HexaneCH3CNEtOAc 5:5:2
cyclohexaneMeOHH2O 10:9:1
Steroids
Enramycins A and B (Fig. 6)
Diterpenes (Fig. 5)
Massoia lactones (Fig. 5)
(72)
(62)
(59)
(61)
HeptaneEtOAcMeOHH2O 1:1:1:1
CCl4MeOHH2O (0.01 M KHPO4 pH7)
CCl4CH2Cl2MeOHH2O 5:5:6:4
Oxysporidinone (Fig. 20)

Niddamycins (Fig. 5)
9-Dihydro-13-acetylbaccatin III
(Fig. 4)
(73)
(58)
(57)
CHCl3EtOAcMeOHH2O 12:8:15:10
EtOAcCCl4MeOHH2O, 1:6:4:1
Pristinamycins (Fig. 3)
Liensinine alkaloids
(55)
(44)
CHCl3MeOHH2O 1:1:1
Coloradocin, 2-norerythromycins,
pentalenolactone (Fig. 12) and
ascelacins (Fig. 13)
(58, 63,
7478)
CHCl3MeOHH2O 7:13:8
Siderochelin (Fig. 20)
(63)
CHCl3MeOHH2O 4:3:3
1,3-Dimethylisoguanine (Fig. 20)
(79)
CHCl3MeOHH2O (0.5%HBr) 5:5:3
Michellamines (Fig. 14)
(80)
n-HexaneEtOAcCH3CNMeOH 5:4:5:2
Triterpene acetates (Fig. 21)
(81)
(continued)
232
Table 4
(continued)
Solvent system
Compounds
References
n-HeptaneC6H6IPAacetoneH2O 5:10:3:2:5
2-Norerythromycins (Fig. 12), and (77, 82)

6-O-methylerythromycin metabolites (Fig. 15)
n-HexaneEtOAcH2O 3:7:5
2-Norerythromycins (Fig. 12)
(77)
EtOAcEtOHH2O 3:1:2
Dorrigocins (Fig. 16)
(83)
EtOAcMeOHH2O
Halishigamides (Fig. 17)
(84)
iso-AmOHn-BuOHn-PrOHH2OHOAc
t-Bu2S 32:48:40:120:1:4
n-BuOHacetoneH2O ~3:1:4
t-BuOMeTHFH2O 2:2:3 (Et3N, HCl)
Tunichromes (Fig. 18)
(85)
Kaempferol glycosides
Harmine, Harmaline (Fig. 2)
(2)
(46)
Et2OMeOHH2O (phosphate buffer

pH 7) 3:1:2
Et2OH2O (Na-phosphate buffer pH 7.57.2)
Giberellins (Fig. 18)
(67)
Liensinine alkaloids (Fig. 2)
(43)
n-BuOHH2O (0.01 N HCl)
Tetracyclines (Fig. 19)
(86)
diverse systems where the activity is all in the aqueous phase of one
and in the organic phase of the other. Now two very simple partitions
will achieve a high level of purification prior to any chromatography. The approach to the ubiquitous HEMWat [hexaneethyl
acetate (EtOAc)methanol (MeOH)water] system, for example,
is to assume that for organics of medium polarity, hexane and water
will be poor solvents, and EtOAc and MeOH good solvents, and
that the lower phase will consist mainly of MeOH and water,
whereas the upper phase will consist mainly of hexane and EtOAc.
Hence if, in the HEMWat-0 (1:1:1:1) system, the desired compound
favors the upper phase, it can be displaced toward the lower, by
increasing the proportion of hexane or MeOH. Increasing the
proportion of MeOH has its limits, particularly in the ChMWat system,
as at some level the system will become monophasic.
Although it is most common, it is not necessary to maintain
the same SS as the mobile phase throughout the chromatography
run. However, preferably any change should not drastically upset
the equilibrium between the two phases. Jia et al. (26) used a stepwise gradient elution though a series of closely related HEMWat
systems to effectively separate six bufadienolides (Fig. 1) from the
Chinese traditional medicine, Chan_Su. They loaded and initially
ran the CCC with HEMWat 2:3:1:2 and the lower phase mobile
for 200 min, then changed the mobile phase to the lower phase of
HEMWat 8:12:5:8 for a further 200 min, and finally to the lower
phase of HEMWat 10:15:8:10 for a further 300 min. A more daring
233
gradient system was used by Liu et al. (27) in the separation of 13

compounds, predominantly flavonoids, from the leaves of Apocynum
venetum in a one-step 90 min chromatography. The column was
charged and loaded with HEMWat 3:7:4:9 and run with the upper
phase mobile. After 30 min, the mobile phase was changed to the
upper phase of EMWat 5:2:5, and after a further 30 min to the
upper phase of BuMWat 5:1:5 (27). Such a radical gradient system
represents a certain degree of faith, as calculated partition coefficients
cease to be translated with the accuracy normally expected and can
certainly not be used to predict the exact position of elution of a
particular solute.
In choosing the SS, it is necessary to avoid those, which form
an emulsion. A useful practical test is to shake well together approximately 1 mL of each of the two phases, and allow the mixture to
separate under gravity. The separation should be complete in 5 s.
3.3. Prediction of
Solvent Systems and
CCC Chromatograms
Octanolwater partition coefficients have been calculated for

chemical structures with high accuracy for decades by pharmacologists and medicinal chemists to predict the ability of compounds to
cross-biological membranes. The calculation of partition coefficients for a given structure in the multicomponent mixtures used
in CCC is a more complex operation. However, Minceva et al.
(28) have shown that the COSMO-RS program developed by
Klamt (29) will provide good predictions of partition coefficients
of a wide variety of compounds, including the GUESS mix, in both
the HEMWat and the very similar ARIZONA (HepEMWat) systems (30). This work is an extension of previous semiempirical
methods that have been proposed. It is advantageous to measure
the partition coefficient of the target compound in a few select
solvent systems in order to predict its partition coefficient in a large
number of solvent systems of the same components. The HEMWat
solvent system family seems particularly suited to this type of prediction methodology. It has been proposed that the K for a compound tested in one HEMWat SS may be predicted for another by
the simple formula log Kb = log Ka + 0.16(b a), where the variable
a is the number of the SS in which K is known, and b the number
of the SS to be predicted (31). Han et al. (32) have proposed a
mathematical model by which the relative proportion of methanol in
an HMWat or HEMWat SS required to give a K = 1 can be calculated
once the K value for the compound is measured in HEMWat
1:1:1:1 (=HEMWat 0). Dubant et al. (33) propose the generation
of a 3D partition coefficient map which empirically measures partition coefficients in eight different SS of the same components.
A seven component subset of the GUESS mix plays a key role
in the validation of a novel approach to modeling CCC described
by Guzlek et al. (34). The calculations are based on the mass balance
equations derived from a theoretical series of identical continuousstirred tank reactors containing a two-phase solvent system.
234
Once the partition coefficients of the analytes are calculated or

measured, the next theoretic step is to predict the chromatographic
separation. Mathematical modeling to predict the outcome of a
countercurrent distribution (CCD) chromatogram was developed
as far back as 1941 by Martin and Synge (35). Recent computational approaches to CCC have sought to apply mathematical
modeling to isocratic flow modes as well as elution-extrusion, dual
flow, and co-current modes. To this end, a universal CCC modeling
based on CCD has been developed by de Folter, Wood, and
Sutherland (CCC3 software) (3638). The preEEdiCCCt software
for the prediction of EECCC chromatograms on volumetric
and K-based scales has also been developed very recently (39).
Nonequilibrated methods, such as pH-zone refining and stepgradient CCC, have so far eluded mathematical modeling.
The importance of partition coefficients (see Note 2) to both
describe and predict chromatographic behavior in CCC has been
accentuated by the recent development of reciprocal symmetry
plots. The GUESS mix standard compound mixture was instrumental in demonstrating the utility of this method of plotting CCC
chromatograms (40).
3.4. pH-Zone Refining
Chromatography
Ionizable compounds usually have very different partition coefficients

in their charged versus their neutral form. Thus, when working
with such compounds, it is advisable to be sure that these compounds are maintained in the same ionization state throughout the
chromatography, and that this has been taken into account when
the SS is being chosen. The pH can be controlled by including
small amounts of an acid or a base in the SS or by including low
concentration buffers as the aqueous component. A good alternative for ionizable compounds is to use the pH-zone-refining
technique, a partition ion-exchange displacement method developed
by Ito (41). In any of these approaches, it is important to take into
account the effect on the solubility of the load sample. The amount
of material, which can be successfully chromatographed, is determined
by this solubility (and of course by the differences in partition
coefficients of the components). Typical loads for a 300 mL coil
are in the order of 200 mg but this can vary by almost an order of
magnitude in either direction dependent on solubility.
Over the last 15 years, Ito has demonstrated the use of CCC in
a semidisplacement mode for ionizable compounds. pH-zone
refining chromatography uses a two-phase aqueous/organic system,
in which each phase is modified with an ion-pairing reagent. Itos
nomenclature is to regard the normal displacement mode as that
with the aqueous phase stationary, and the reverse displacement
mode as that with this phase as mobile. He refers to the modifier
in the stationary phase as a retainer and that in the mobile phase
as a displacer. The optimal concentration of each modifier is different
for the two modes. For example, in the separation of Crinum moorei

O
OH
235
OH
O
N
R
Crinine R=H
OMe
Crinamidine
Powelline R=OMe
O
N
O
OH
Liensinine R1 = H, R2 = CH3
Isoliensinine R1 = CH3, R2 = H
Neferine
R1 = R2 = CH3
OR1
OR2
O
H3CO
O
N
H3CO
N
O
Nuciferine R= CH3
N-Nornuciferine R = H
Roemerine
H3CO
H3CO
N
H
Harmine
N
H
Harmaline
Fig. 2. Structures of Crinum moorei, Nelumbo nucifera, and harmane alkaloids.
alkaloids, in a t-butyl methyl ether/water system, in the normal

displacement mode the aqueous stationary phase was modified with
10 mM HCl and the mobile organic phase with 10 mM triethylamine,
whereas in the reverse displacement mode the same modifiers were
used at half this concentration. In each case, a 3 g crude mixture of
the three alkaloids, crinine, powelline, and crinamidine (Fig. 2) were
completely separated on a 300 mL capacity Ito Coil instrument;
however, the reversed-phase mode was complete in 150 min,
whereas the normal phase required almost 400 min (42). This technique is really taking opportune advantage of the large difference
in partitioning of the same molecular skeleton in charged and
uncharged forms. As a result, unlike typical CCC, pH-zone-refining
236
results in elution of the analytes as rectangular peaks, with impurities

concentrated at the interfaces between peaks. Ito states that these
two modifiers, HCl and triethylamine have proved successful for all
of the separations of organic bases that he has undertaken, but that
in the separation of acids the most versatile modifiers are trifluoroacetic
acid and ammonium hydroxide. However, other acids have sometimes
been more desirable (42). This mode can really be considered as a
hybrid between ion-exchange chromatography and CCC and consequently it is noticeable that the loading capacity of most pHzone
refining chromatographies is greatly enhanced over typical partition
chromatography. This is exemplified by the separation of the alkaloids,
liensinine, isoliensinine, and neferine (Fig. 2) from the seeds of
Nelumbo nucifera Gaertn. Wu et al. (43) reported preparative
separations in two conventional CCC systems; light petroleum
EtOAcCCl4CHCl3MeOHH2O, 1:1:4:4:6:2 and EtOAcCCl4
MeOHH2O, 1:6:4:1 on four coil 1.6 L capacity J-type instrument.
The first system was used for small scale and a 1,102 mg load was
separated into a total of 545 mg of the three alkaloids with purities
of >95% (assay unspecified). Liu and colleagues (44) used a gradient
pH buffer (sodium hydrogen phosphate from pH 7.5 to 7.2) as the
mobile phase with diethyl ether as the stationary phase in a 260 ml
capacity instrument. From a load of 200 mg of crude extract they
isolated a total of 142 mg of the three alkaloids in over 98% purity.
Two more striking examples of the capacity difference between
pH-zone refining and conventional HSCC is offered by Zheng
et al. (45) and Wang et al. (46). The former separated the aporphine
alkaloids, nuciferine, N-nornuciferine, and roemerine (Fig. 2) from
the leaves of N. nucifera Gaertn. by conventional HSCC in CCl4
CHCl3MeOHH2O (0.1 N HCl) 1:3:3:2 and obtained a total of
33 mg from 120 mg of crude extract. Whereas in a pH zone refining
mode with pet etherEtOAcMeOHH2O 5:5:2:8 with 10 mM
Et3N as the retainer and 5 mM HCl as the displacer, they were able
to load 4 g on the same instrument and obtain 1,236 mg of
the three alkaloids. In every case, the product was >98% pure and the
chromatograms shown suggest that the conventional HSCC may
have been closer to maximum capacity. The latter group separated
the crude harmane alkaloid extract of Peganum harmala on a
236 mL capacity J-type instrument in a methyl t-butyl etherTHF
H2O system with 10 mM Et3N as the retainer and 5 mM HCl as
the displacer, and obtained 554 mg of harmine and 325 mg of harmaline (Fig. 2) from 1.2 g of extract (46).
3.5. Physical Aspects
of Operation
All of the CCC instruments are effectively closed systems. It is not

necessary to locate the actual instrument in an exhaust hood; however, the solvent will be pumped from reservoirs, and the eluent is
usually collected in a fraction collector. Since almost all systems
involve volatile organic solvents, it is advisable to locate these
peripherals in a hood. The pump must be capable of delivering
237
between 2 and 5 mL/min, and should not produce large pulses.

A typical 3-way injection valve is required, and the sample can be
loaded in any volume from 1 to 10 mL. It is of paramount importance
that the sample be completely dissolved. To avoid any possibility of
salting out it is common to load the sample in a mixture of the two
phases. The machine should be fully loaded and at least the initial
parts of the instrument should be equilibrated and rotating before
the sample is loaded.
3.6. Use of the Ito Coil
Planet Centrifuge
When using the Ito coil, the researcher is presented with the making a choice of 3 twofold variables.
(a) The question of which phase to select as the stationary phase,
i.e., which phase is used to fill the column.
(b) The choice of the inlet tube, either the head or the tail of
the column.
(c) The question of which direction to spin the column, i.e., to
have the Archimedean force directed to the inside or the outside of the spiral.
Two of the eight (23) possibilities will usually work well, two
poorly and the other four will result in no retention of stationary
phase. A more detailed description of the binary choices in CCC
instrument design and operation has been recently published (47).
The tubings to the columns are labeled head and tail, and
spin directions as forward and reverse. It is advisable to fill the
column with the stationary phase while it is spinning with an
Archimedean force against the fill. This ensures that the column is
filled without any vapor blocks. The column does not have to be
spun at normal running speed as this may create balancing problems.
When the column is filled with stationary phase, it should be spun
at 8001,000 rpm while pumping the mobile phase. It is possible
to introduce the sample load with the solvent front of the mobile
phase, but in a new system this is usually unwise. The system can
be tested to ascertain the displacement of stationary phase before
introducing the sample. In the better systems only about 10% of
the stationary phase is displaced before breakthrough of the mobile
phase. After that only the mobile phase is eluted from the column.
It is useful to keep in mind the theoretical shape of the elution
curves. Although this varies with the nature of the solute (48), and
to a lesser extent the SS, for most natural products the following
generalization applies. Components with partition coefficients
strongly favoring the mobile phase will be eluted very early in chromatography, and will be in sharp peaks. As the chromatography
continues, the peaks eluted with the mobile phase broaden. In
early use of HSCCC, it was common to pump two to three column
volumes of mobile phase and then to reverse the direction of spin,
and displace the stationary phase either by continuing to pump
mobile phase or with a stream of nitrogen. The stationary phase is
238
collected in fractions as separations will have been effected but the

compounds not yet eluted. Today, it is more common to change
the solvent being pumped into the column to the stationary
phase and to do this immediately after a single column volume of
mobile phase has been eluted from the column. This protocol
is referred to as elution extrusion CCC (EECCC) (49) (see Note 3).
Components eluted with the stationary phase will also come as
sharp peaks, and the whole process is more environmentally favorable
as the entire load is eluted from the column in just two column
volumes and good separation is achieved for components with a
wide range of polarities.
3.7. Use of the
Centrifugal Partition
Chromatograph
With this mode of CCC the choices are more straightforward for
the novice. The only choice that needs to be made in running the
instrument is which phase is to be the mobile phase. The CPC
instruments come with a conveniently labeled switch, ascending
descending, and this can be mentally placed in the old droplet
format, in which the upper phase mobile will be ascending and
the converse. One does not have to carefully estimate counterbalances. CPCs have neoprene or ceramic seals, which require replacement after extensive operation, the ceramic ones lasting much longer
than the neoprene ones, but are more expensive. These instruments
are loaded with a spin speed of approximately 200 rpm for the
200 mL rotor and 900 rpm for the 1 L instruments. A flow rate of
between 1 and 5 mL/min is appropriate for the 200 mL instrument,
dependent of the relative densities of the two phases. However, 1 L
instruments are run with flow rates between 20 and 30 mL/min,
provided that the pressure on the column does not exceed 800 psi.
These instruments are relatively quiet and stable.
For all instruments certain parameters are absolutely critical,
such as direction of spin, direction of flow and choice of stationary/
mobile phases and the separation time (differential density) of the
phases. Others are less critical but nonetheless have a significant
effect on performance. The optimal adjustment of flow rate, temperature, rotational speed, and sample loading in a 240 mL hydrodynamic (J-type) CCC instrument was investigated by Friesen and
Pauli (50) utilizing the complete GUESS mix in HEMWat 4:6:4:6
(=HEMWat +3).
3.8. Detection
In principle any of the detection systems commonly used in

chromatography can be used to monitor the analytes in the eluate.
In practice, however, UV detectors give esthetically displeasing
traces as the miniscule leakage of the stationary phase results in a
spiked curve. Evaporative light scattering detectors (ELSDs)
have proven satisfactory (51) and advantageous in not requiring a
chromophore (see Note 4). However, since ELSD is a destructive
form of detection, unless CCC is performed in a purely analytical
manner at small scale and flow rates, it will require flow splitters
to accommodate the typical preparative CCC. In practice, with
239
biologically active compounds, it is usually convenient to simply

collect CCC fractions and subsequently assay these, off-line, for
activity, and/or by TLC.
Recently, practical implementations of online MS detection
have been reported by practitioners of CCC. Han et al. (52) performed ESI quadrupole time-of-flight (QTOF) mass spectrometry
while doing a bioassay-guided discovery of apoptosis inducers from
gamboge. In the group of Winterhalter et al. (53), Bougainvillea
glabra betacyanins were detected online by coupling preparative
ion-pair high-speed countercurrent chromatography with ESI-MS.
In instances of biological activities for which LC-compatible
assay methods are available, their adaptation to CCC is fully feasible as shown very recently by Lim et al. (54), who coupled CCC of
licorice (Glycyrrhiza glabra) extracts with an online assay for antioxidant activity in order to identify the active principles.
3.9. Examples of CCC
Separations of Natural
Products
The following few examples from our laboratories (see Note 5)

and the literature have been chosen to represent the power of the
method. In each case, baseline or near baseline separation of two
close structurally related congeners has been obtained. Each represents a considerable challenge, in particular when approached by a
non-CCC separatory method.
3.9.1. Separation
of Pristinamycins
The pristinamycins (Fig. 3) are an unusual complex of antibiotics

in that they consist of two pairs of peptolide antibiotics very
closely related within the two pairs but with virtually no structural relationship from one pair to the other. Pristinamycins IA
and IB differ only in the degree of N-methylation of a 4-aminophenylalanine moiety while pristinamycins IIA and IIB differ only
in that IIA has a 2,3-dehydroproline moiety where IIB has a proline. Thibaut et al. (55) were able to achieve baseline separations
between IIA and IIB with a system comprising CHCl3EtOAc
MeOHwater (12:8:15:10) on a triple planetary coil instrument
at 1,400 rpm with the upper phase mobile. Pristinamycins IA and
IB were best separated with a system in which the same components were in the ratio 6:4:8:1, where the last component was
formic acid to control the pH but of otherwise unspecified
strength.
3.9.2. Separation of Taxol

and Cephalomannine
The anticancer agent, Taxol, can be obtained from a number of

Taxus species, but invariably it occurs with sizeable amounts of
the congener, cephalomannine (Fig. 4). These complex diterpenes
differ only in the nature of the amide carboxylic acid attached to
the amine of the phenylisoserine side-chain. In the case of Taxol,
this is a benzoic acid moiety whereas in cephalomannine it is a
tiglic acid group. These two impart very little selective polarity
to the two natural products and separation of them is notoriously
difficult. Almost baseline separation of a small sample (6.1 mg) was
achieved however by Chu et al. (56) using a system of hexane
240

O
OH
N
O
H
O
O
N
N
O
Pristinamycin IIA
O
OH
N
O
H
O
O
N
Pristinamycin IIB
OH
O
N
NH
N
O
N
H
H
N
O
O
O
N
N
N
Pristinamycin IA (R = Me) and IB (R = H)
Fig. 3. Structures of pristinamycins.
O
O
O
O
O
O
O
HO
OH
HO
HO
O
O
O
O
HN
R
Taxol R =
O
OH O
OH O
O
O
Cephalomannine R =
9-Dihydro-13-acetylbaccatin III
Fig. 4. Structures of Taxol, cephalomannine, and 9-dihydro-13-acetylbaccatin III.
241
EtOAcMeOHethanolwater (10:14:10:2:13) with the aqueous

phase mobile. In this system, Taxol had a partition coefficient of
1.8 and cephalomannine of 1.42. In the isolation of 9-dihydro-13acetylbaccatin III (Fig. 4) from Taxus canadensis, the taxanes were
separated by HSCCC with isooctaneEtOAcMeOHwater
(7:3:6:4) with the organic phase mobile (57).
3.9.3. Separation
of Niddamycins
The 16-membered antibacterial macrolide niddamycin complex

(Fig. 5) is produced by Streptomyces djakartensis as a mixture of
aliphatic esters of the 3-hydroxyl, the secondary alcohol on the
neutral sugar mycarose. Niddamycin A1 has a butyryl ester, whereas
niddamycin B has the isovaleryl ester at this position. In addition,
another congener, niddamycin F is similar to niddamycin A1 except
that the 10, 11 olefinic bond in the macrolide ring of A1 is fully
reduced in niddamycin F. Chen et al. (58) achieved baseline separation of a 200 mg sample of all three niddamycins on an Ito coil
in a system of carbon tetrachloridemethanol0.01 M aqueous
phosphate buffer at pH7 in the ratio 2:3:2. With the aqueous phase
mobile niddamycin A1 was eluted first followed by niddamycin B,
while niddamycin F was retained, and was recovered from fractions
of the stationary phase when it was pumped from the column.
3.9.4. Isolation of
Kaurenoic and Polyalthic
Acids from Oleoresin
A nonaqueous system, HAcE, 5:5:2, was used to isolate two diterpenes, kaurenoic acid, and polyalthic acid (Fig. 5) from the oleoresin of Copaifera glycycarpa, which is used as a folk medicine in the
Brazilian amazon (59). From 600 mg of resin, 35 mg of kaurenoic
acid and 150 mg of polyalthic acid were obtained using a two-bobbin AECS Quattro instrument with a capacity of 234 mL per bobbin. With the upper phase mobile and pumping at 3 mL/min the
retention of stationary phase was 92%. The two diterpenes showed
moderate activity inhibiting multiple-drug resistant transporters.
3.9.5. Purification of
Tanshinones from Tanshen
Tanshinones are regarded as major bioactive constituents of the

traditional Chinese medicine Tanshen, an extract from the rhizome
of Salvia miltiorrhiza Bunge. Wu et al. (60) determined the partition
coefficients, by HPLC, of tanshinone I and tanshinone IIA (Fig. 5)
in several HEEtWat systems and chose 8:2:7:3, in which the target
compounds had K values of 0.56 and 1.42, respectively. From 50 mg
of crude extract, 1.5 mg of tanshinone I and 7.2 mg of tanshinone
IIA were obtained in >94% and >98% HPLC purity, respectively,
using a Tauto TBE-300A instrument. These scientists also analyzed
the compositions of the equilibrated upper and lower phases by
GC in order to obviate the need for pre-equilibration of solvent.
3.9.6. The Isolation

of Massoia Lactones
The bark of Cryptocarya massoy is extracted to produce massoia oil,

which is used extensively as a flavoring agent in foods or as a fragrance
in perfumes or massage oils. The main components imparting this
fragrance are a series of unsaturated aliphatic delta lactones. These
have been extracted from the oil and separated by hydrostatic CCC
242

O
10
CHO
11
N
HO
O
H3CO
O
O
OH
OH
OR
O
Niddamycin A1
R = COCH2CH(CH3)2
Niddamycin B
R = COCH2CH2CH3
Niddamycin F = 10,11-Dihydroniddamycin A1
O
H
H
H
COOH
COOH
Kaurenoic acid
Polyalthic acid
O
O
O
Tanshinone I
Tanshinone IIA
(R)-(-)-C-10 Massoia lactone n = 1

Fig. 5. Structures of niddamycins, Copaiba oil diterpenes, tanshinones, and massoia lactones.
on a Kromaton 1 L instrument with an SS consisting of cyclohexane

MeOHH2O, 10:9:1, run in the ascending mode. The three lactones
(R)-()-C-10 massoia lactone, (R)-()-C-12 massoia lactone, and
(R)-()-C-14 massoia lactone (Fig. 5) had K values of 0.89, 1.41
and 2.46, respectively, in this SS, and a 10 g load yielded 3.17 g,
357 mg, and 21 mg in that order, all of >96% HPLC purity (61).
3.9.7. Separation
of Enramycins A and B
Perhaps no example portrays the exquisite sensitivity of the method

as the separation of enramycins A and B (Fig. 6). These two congeneric peptolides have MWs over 2,000, each have two guanidino
groups, a lysine and an aminol. Hence, they are very basic and very
fragile. They differ by a single aliphatic methyl group at the end of a
fatty acid chain. However, Inoue et al. (62) were able to separate a
15 mg sample of the antibiotic complex and obtain 4.3 mg of enramycin A and 5.9 mg of enramycin B with an RS value of 2.9 in a simple
HSCCC system of HeBuWat (0.05% TFA) 7:43:50 with only a 54%
retention of stationary phase using a Pharma-Tech CCC-1000.

R
O
HO
243
Tirandamycin A R = H
Tirandamycin B R = OH
NH
O
O
CH2
CO2H
H
CH3
OR
CH3
OR'
HO
CH3
X=
O
CO2H OH
CO2H
CH3
Squalestatin A R = Ac, R' = X

B R = H, R' = X
C R = Ac, R' = H
R
O
HO
O
O
O
HN
HN
N
H
NH
HN
O
NH
HO2C
N
H
NH2 O
OH
HN
NH
O
NH
O
HN
Cl
NH2
O
OH
O
NH
O
NH
NH
O
HN
NH
O
HO
OH
HN
NH
Cl
HO
NH
H
N
N
H
H
N
OH
OH
OH
O
NH
HN
Enramycins A (R = H) and B (R = CH3)
Fig. 6. Structures of tirandamycins, squalestatins, and enramycins.
4. Notes
1. Targeted toward high sample diversity, GUESS is a practical
approach for the prediction of CCC distribution constants, K
values, by standard thin layer chromatography (TLC). It allows
a major reduction in workload by direct use of routine TLC
information. As SSs usually involve several solvents and repeated
description of them becomes tiresome, a simplified abbreviation
set has been developed (21) both for the individual components
244

OR
R'O
O
Arizonin A1
R = Me
Arizonin B1
Arizonin C1
R=H
R = Me
R = R = Me
OR
R = H
R'O
O
O
OCH3
OH
Arizonin A2
R = Me
R = H
Arizonin B2
R=H
R = Me
Arizonin C2
R = R = Me
OMe
OH
O
OH
O
O
O
HO
HO
OH
MeO
O
H2N
Concanamycin A (R = Me) and Concanamycin B (R = H)
Fig. 7. Structures of arizonins and concanamycins.
H
N
OH
COOH
R
Indole-3-acetonitrile R = -CH2CN
Abscisic acid
Indole-3-acetamide R = -CH2CONH2
Indole-3-acetic acid R = -CH2COOH
Indole-3-butyric acid R = -(CH2)3COOH
Indole-3-carboxylic acid R = -COOH
Fig. 8. Structures of auxins.
and, by combination, for the systems (Tables 13). GUESS has

been shown to be readily applicable to natural product
purification necessary for drug discovery, bioassay-guided
fractionation, and metabolome analysis.
245
OH
O
H
HO
H
O
O
H
OH
Australifungin
O
O
O
O
OH
O
O
O
C
OH
OH
O
O
OH
O
OH
OH
O
E
OH
D
Phomopsolides
Fig. 9. Structures of australifungin and phomopsolides.
O
O
O
O
OH
HO
OH
Isotrichoverrin A (R = H) and Isotrichoverrin B (R = H)
Fig. 10. Structures of trichoveroids.
2. A partition coefficient or a distribution coefficient is the ratio of

concentrations of a compound in the two phases of a mixture of
two immiscible solvents at equilibrium. Thus, these coefficients
are a measure of differential solubility of the compound between
these two solvents. Generally, one of the solvents chosen is
water while the second is a hydrophobic solvent, e.g., octanol.
Therefore, the partition or distribution coefficient is a measure
of how hydrophilic or hydrophobic a chemical substance is.
3. The elutionextrusion method has two steps: the first step is a
regular CCC chromatogram. Next, the stationary phase
246

O
O
O
O
O
NH
Bu 2313 B
O
O
N
N
H
N
5-N-Acetylardeemin
Fig. 11. Structures of Bu 2313 B and 5-N-acetylardeemin.

H
H
H3CO
O
O
H
OH
COOH
O
HO
O
O
H
O
O
O
O
Pentalenolactone
Coloradocin
O
OH
R'
OH
HO
O
O
O
R
OH
O
2-Norerythromycin A
R = OMe
R = OH
2-Norerythromycin B
R = OMe
R = H
2-Norerythromycin C
R = OH
R = OH
2-Norerythromycin D
R = OH
R = H
Fig. 12. Structures of coloradocin, pentalenolactone, and 2-norerythromycins.
247
H
N
OH
O
O
NH
N
H
HN
O
HN
O
H3C
HN
N
H
H2N
O
OH
Aselacin A
R=
CH3
O
OH
Aselacin B
R=
CH3
O
O
Aselacin C
R=
CH3
Fig. 13. Structures of ascelacins.
OH
OH
NH
HN
OH
OH
OMe
OMe
OH
OMe
OH
HO
OMe
OH
HO
NH
OH
Michellamine A
Fig. 14. Structures of michellamines.
NH
OH
Michellamine B
248

O
H3C
CH3
HO
OCH3
H3C
H3C
CH3
OH
R2
CH3
HO
H2
C
H3C
CH3
OCH3
CH3
CH3
OH
CH3
Clarithromycin (6-O-Methylerythromycin) R1 = R2 = H
(14R)-14-Hydroxyclarithromycin R1 = OH, R2 = H
15-Hydroxyclarithromycin R1 = H, R2 = OH
Fig. 15. Structures of 6-O-methylerythromycin metabolites.
O
HN
OH
OH
COOH
O
OCH3
Dorrigocin A
O
HN
OH
OH
COOH
O
OCH3
Dorrigocin B
Fig. 16. Structures of dorrigocins.
containing the separated retained solutes is extruded out of the

column in a continuous way using the liquid stationary phase.
4. Evaporative light scattering (ELS) detection can offer distinct
advantages over ultravioletvisible (UV/Vis) and refractive
index (RI) detection for particular applications, and is complementary to mass spectrometry. The technique is a powerful
tool for detecting any sample that is less volatile than the
mobile phase, irrespective of the optical properties of the compounds of interest.
5. The authors gratefully acknowledge the dedicated work of our
academic and industrial coworkers at UIC, Abbott Laboratories,
Phytera, and Ecopia, who have contributed so much to our
CCC research and publication portfolios.
OHC
249
O
N
OCH3 O
OCH3 O
O
O
NH2
N
O
N
O
NH2
OCH3
HN
O
O
OCH3
O
N
NH2
CO2CH3
CONH2
CONH2
NH2
CO2CH3
N
O
OCH3
OCH3
Fig. 17. Structures of halishigamides.
OH
OH
OH
OH
HO
HN
H
N
O
OH
NH2
OH
OH
Tunichrome A1 (R = H) and tunichrome B1 (R = OH)

O
HO
OH
OH
OH
Gibberellic acid
Fig. 18. Structures of tunichromes and gibberellic acid.
250
OH
N
OH
NH2
OH
OH
OH
Tetracycline (R = H) and Oxytetracycline (R = OH)
Fig. 19. Structures of tetracyclines.
O
OH
O
OH
OH
N
Oxysporidinone
NH
HO
N
H2N
N
H
OH
1,3-Dimethylguanine
Siderochelin A
Fig. 20. Structures of oxysporidinone, siderochelin, and 1,3-dimethylisoguanine.
CH2
CH2
H3C
H3C
H
CH3
CH3
CH3
CH3
Taraxasterol acetate
O
O
H3 C
CH3
CH3
H3C
CH3
CH3
H
CH3
CH3
H
CH3
-Amyrin acetate
H3C
CH3
H3C
Lup-20(29)en-3b-ol acetate
CH3
H
CH3
CH3
CH3
Taraxasterol acetate
Fig. 21. Structure of triterpene acetates.
-Amyrin acetate
CH3
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Chapter 9

Separation of Natural Products by Countercurrent

J.B. McAlpine et al.

Separation of Natural Products by Countercurrent Chromatography

Kaempferol glycosides from Pouteria obovata

Gamabufotalin R = OH, R' = R" = H

High-speed CCC (HSCCC) instruments became commercially

J.B. McAlpine et al.

covers phytochemicals very comprehensively, and Sutherland and

Separation of Natural Products by Countercurrent Chromatography

Chromatograph is the rotating seal, which must remain solvent

J.B. McAlpine et al.

When commercial instruments of this design become readily available,

Separation of Natural Products by Countercurrent Chromatography

3.1.2. The Development

J.B. McAlpine et al.

As the GUESS methodology has become more widely accepted,

In the course of natural product isolation, the chemist is often

Separation of Natural Products by Countercurrent Chromatography

The equivolumetric mixture, HEMWat 0, represents the portal SS of the family,

J.B. McAlpine et al.

relatives, which may differ only by one or two methylenes, the

Separation of Natural Products by Countercurrent Chromatography

Liensinine alkaloids (Fig. 2)

Arizonins, concanamycins (Fig. 7)

n-HexaneEtOAcMeOHH2O (0.01 N H2SO4)

Taxol and cephalomannine (Fig. 4) (56)

Trichoveroids (Fig. 10)

Bu 2313 B (A tetramic acid),

Oxysporidinone (Fig. 20)

Siderochelin (Fig. 20)

1,3-Dimethylisoguanine (Fig. 20)

CHCl3MeOHH2O (0.5%HBr) 5:5:3

Michellamines (Fig. 14)

Triterpene acetates (Fig. 21)

J.B. McAlpine et al.

2-Norerythromycins (Fig. 12), and (77, 82)

2-Norerythromycins (Fig. 12)

Dorrigocins (Fig. 16)

Halishigamides (Fig. 17)

Tunichromes (Fig. 18)

Et2OMeOHH2O (phosphate buffer

Giberellins (Fig. 18)

Liensinine alkaloids (Fig. 2)

n-BuOHH2O (0.01 N HCl)

Tetracyclines (Fig. 19)

Separation of Natural Products by Countercurrent Chromatography

gradient system was used by Liu et al. (27) in the separation of 13

Octanolwater partition coefficients have been calculated for

J.B. McAlpine et al.

Once the partition coefficients of the analytes are calculated or

Ionizable compounds usually have very different partition coefficients

Separation of Natural Products by Countercurrent Chromatography

Fig. 2. Structures of Crinum moorei, Nelumbo nucifera, and harmane alkaloids.

alkaloids, in a t-butyl methyl ether/water system, in the normal

J.B. McAlpine et al.

results in elution of the analytes as rectangular peaks, with impurities

All of the CCC instruments are effectively closed systems. It is not

Separation of Natural Products by Countercurrent Chromatography

between 2 and 5 mL/min, and should not produce large pulses.

J.B. McAlpine et al.

collected in fractions as separations will have been effected but the

In principle any of the detection systems commonly used in

Separation of Natural Products by Countercurrent Chromatography