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Alain P. Foucault Laboratoire de Bioorganique et Biotechnologies Ecole Nationale Superieure de Chimie de Paris 11, rue Pierre et Marie Curie 75231 Paris Cedex 05 France
Countercurrent chromatography (CCC) generally refers to supportfree liquid-liquid chromatography with two immiscible liquids prepared by mixing two or more solvents or solutions. An instrument keeps one liquid stationary while the other liquid is pumped through it, and the chromatographic process occurs between the two liquid phases. Modern CCC originated with the pioneering studies of Ito, Nunogaki, and co-workers in Japan ( I ) . They constructed an apparatus designed to differentiate particles in suspension or solutes in solution in a solvent system subjected to a centrifugal acceleration field. This device-the coil planet centrifuge-opened the way to the fruitful research conducted by Ito in the United States, which has been devoted primarily to the study 0003-2700/91/0363-569A/$02.50/0 0 1991 American Chemical Society
and realization of many types of CCC apparatus, some of which are commercially available. Other groups also have been working with CCC, and eight to ten instruments are now commercially available. CCC and its historical development were previously reviewed in this JOURNAL (2).In this article, we will discuss the theory of CCC and compare it with HPLC, describe commercially available and prototype instruments, and emphasize recent advances that make the technique more efficient and faster. CCC and HPLC CCC and HPLC are similar in several respects. Both are based on the same fundamental mechanism, the partitioning of solutes, which determines the distribution of the molecules in the various phases. Both techniques have the same goal-to differentiate solutes in a mixture in order to isolate one or more of them.
The manner in which this goal is achieved is also similar. An apparatus keeps one phase stationary as a second phase moves through it. Solutes are collected as they elute. Finally, if one wishes to use CCC or HPLC preparatively, economics be come important. One must deal with the costs incurred in purification versus the market price of the pure compound recovered. Except for different separation devices (columns), CCC and HPLC share the same technologies. The same pump, injector, and detector can be used, although minor restrictions can arise in certain cases. Figure 1is a schematic comparison between HPLC and CCC. For convenience, we have chosen a reversedphase chromatographic process. In HPLC (left), the stationary phase is a n organic moiety bonded to silica and solvated mainly by the organic solvent of the aqueous organic mobile phase. The volume ratio of the sta-
ANALYTICAL CHEMISTRY, VOL.
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INSrRUMEN 7ArION
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Figure 1. Schematic comparison between conventional reversed-phase HPLC and reversed-phase CCC. (a) The silica gel surface is shown with a hexyl bonded phase saturated with methanol. (b) This impregnated liquid stationary phase of hexane/methanol (- 5% of the total volume) is in equilibrium with the water/ methanol mobile phase (which occupies the interstitial volume and most of the porous volume, -75% of the total volume). The bulk volume of silica gel, which does not participate in the partition process, occupies 20 - 30% of the volume. (c) Silica gel is eliminated, and a strong gravitational field allows the heavy droplets of water/methanolto flow through the hexane (saturated primarily with methanol) stationary phase. (d) The total volume is used for the partition process, and the ratio V, / V,, depending upon the experiment, is 50:50.Abbreviations: V,, V , , Kat, and Vsi are, respectively, the volumes of stationary and mobile phases, total volume, and volume of solid silica gel.
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tionary phase is only - 5% of the total volume of the column (3), and it will not change significantly as the mobile -phase composition varies. I n CCC (right), the silica is replaced with a strong gravitational field that “frees” 20-30% of the total volume. (The porosity factor, E,, defined as the ratio of the void volume to the total volume of a column, is -0.7 -0.8 for porous silica and -0.35 for solid beads.) Instead of an organic moiety bonded to a solid support, we now simply use a n immiscible organic solvent (e.g., hexane) as the stationary phase; the heavier droplets of the water-organic mixture pass through the stationary phase. The volume ratio of the stationary phase is 5040% for most cases; the actual value depends on the instrument, the experimental conditions, and the composition of the ternary mixture used to prepare the mobile and stationary phases.
CCC: basic systems Ito defined two basic systems of CCC (Figure 2). The first is called the hydrostatic equilibrium system, or HSES. The “column” can be visual570 A
ized as a stationary coiled tube ( r is the radius of the coilJ subjected to a gravitational field, G , which is constant a t any point in the coil. The second system is called the hydrodynamic equilibrium system, or HDES. The “column” can be viewed as a coiled tube rotating along its own axis and subjecJ to a constant gravitational field, G . The resulting
gravitational field + 0 2 r ) varies with the position of the analyzed point in the coil (basically a rotating coil that exhibits an “Archimedean screw” effect). For both cases, the constant gravitational field can be augmented with a revolutionary rotation around an extra axis (Ris the radius for the revolution), which may or may not be parallel to the axis of the coil. As shown in Figure 2, HDES provides better mixing than does HSES, thus favoring the partition step of the chromatographic process. All CCC schemes that have been developed stem from these two basic systems. To improve the efficiency of HSES, the helical column of the original model has been greatly modified to improve t h e partition efficiency; most HSES devices have completely lost their helical appearance. Two well-known examples are the droplet countercurrent chromatograph (DCCC) and the centrifugal partition chromatograph (CPC Model LLN). Various configurations exist for HDES, depending on the relative orientation of the two rotational axes and the ratio of the two radii to the two rotation speeds. Most have been studied by Ito and have been classified as schemes I, L, J, and X, as well as combinations of these. All are configured to allow a rotary seal-free connection with the pump and the detector. HDES devices have r e tained their original helical column configurations; one well-known example based on synchronous scheme J is the high-speed countercurrent chromatograph (HSCCC). Improvements on t h e original technology have provided fast and efficient instruments. Although not yet comparable in speed and efficiency to HPLC, the CCC devices compensate
Figure 2. Two basic CCC systems. (a) The hydrostatic equilibrium system (HSES) consists of a coiled column subjected to a constant gravitational field. The external half of one coil is filled with the heavy mobile phase (dark red) and the internal half is occupied by droplets or clouds of heavy phase falling in the stationary phase. (b) The hydrodynamic equilibrium system (HDES) consists of a coiled column subjected to a fluctuating gravitational field produced by an additional rotation of the coil around its own axis, thus enhancing mixing of the two phases. R is the radius of revolution, r is the radius of the coil, and w is the speed of revolution.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991
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INSrRUMEN 7ArION for these shortcomings with attractive features such as the large capacity of the stationary phase, total recovery of a sample, and low cost of the “packing” (i.e., solvents). CCC: advantages
An advantage of CCC is that the volume ratio of the stationary phase to the total volume is much higher than that of HPLC. This large ratio has two major consequences. First, the capacity of a CCC column is much higher than that of a n HPLC column for the same total volume. Overloading is uncommon in CCC; the limiting factor is the solubility of the sample in both the stationary and mobile phases rather than the amount injected. This makes CCC very attractive for preparative -scale chroma t o g r a p h y , w h e r e it is a l r e a d y competitive and frequently better than preparative -scale HPLC. For the same column volume, one can purify larger quantities with CCC with total recovery (no adsorption) and low operating costs (the cost of the solvents only).
Figure 4. Dual-mode elution in CCC (see text). (Adapted from Reference 6, p. 521 by courtesy of Marcel Dekker, Inc.)
Second, the resolution for two adjacent peaks is
R, = (1/4) (a-1) N1” [ k ; / ( k ; + 1)l (1) where a is the separation factor (It;/ k i ) , I t ; is the capacity factor for the peak 1, and N is the number of theoretical plates. The term I t ; is related to the partition coefficient Kl by
Figure 3. Relationship for two adjacent peaks between the resolution R,, the number of theoretical plates N, and the percent of stationary phase o/oV, in a chromatographic column for HPLC and CCC. (a) For a given N,resolution is better in CCC than in HPLC. (b) CCC requires fewer theoretical plates than HPLC to provide the same resolution. The partition coefficients K,, K2, and the separation factor ct are assumed to be constant. The volume percent of the stationary phase is -5% for HPLC and is usually 50 - 80% for CCC.
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where V, and Vm are the stationaryand mobile -phase volumes, respec tively. Kl is defined (as in HPLC) as the ratio of the concentration of the solute in the stationary phase to the concentration of the solute in the mobile phase. Using these relationships, we can replace It ;in Equation 1 and obtain
The term VJV,, which is always large in HPLC (-13-20), is much
ANALYTICAL CHEMISTRY, VOL. 63, NO.
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smaller in CCC (0.25-1 for a stationary phase occupying 5 0 4 0 % of the total volume). This means that for a given set of separation factors and partition coefficients, the resolution is much higher in CCC for a given number of theoretical plates (Figure 3a). Put another way, it means that the number of plates required to obtain a given resolution is less important in CCC than in HPLC (Figure 3b). As shown in the example in Figure 3, for a = 1.2 and Kl = 1, 1000 theoaretical plates will give R, = 0.1 for HPLC (V, - 7% of the total volume), and R, = 1 for CCC (V, - 65% of the total volume). To obtain R, = 1.5, -185 000 plates will be required in HPLC, whereas 2200 plates will be enough for CCC (Figure 3b).
Efficiency and resolution If we can minimize the smallest physical unit in which the partition process takes place, what effect will this have on CCC? This unit is still
relatively large compared with the cubic-nanometer-scale volume of a silica particle cavity that we find in HPLC. Recent advances have pointed out this smallest physical unit that we can call a physical plate; the goal is to achieve a theoretical plate as close as possible to the physical plate, and to make the latter as small as possible, in order to produce efficient and speedy instruments. Three steps are required to achieve high chromatographic efficiencies. First, the two phases must be mixed until equilibrium is reached, that is, when the ratio of the concentration for a given solute in the two phases is equal to or very close to the partition coefficient K. This step is favored by a large interface area compared with the bulk volume of the two phases. CCC presents essentially the same concerns t h a t researchers encountered with conventional chromatography and initially solved by using pellicular solid beads and, later, very small particles of silica with a high
surface area per unit mass. Next, the two phases must settle into two layers. This is achieved with a strong centrifugal field. The final step is the transfer of the mobile phase to the next physical plate. Of the various parameters that influence efficiency, the flow rate is the most important. HPLC researchers are well aware of this fact; the van Deemter plot [height equivalent to a theoretical plate (HETP) = f(flow rate)] is one of the first relationships they learned. A complete study of van Deemter plots has been published by Armstrong et al. (4), who used a n instrument based on HSES (the CPC Model LLN) to show that van Deemtertype plots exhibit maxima rather than minima. Consequently, the best efficiency is obtained a t high or low flow rates. This observation is significant because analogous flow rates a r e often used for CCC a n d for HPLC; unfortunately, this leads to the worst efficiency. I t is far better to
use higher flow rates, which favor more efficient and faster separations. Ito and Oka have obtained a similar result with an instrument based on HDES (an HSCCC prototype) (5). One must keep in mind that the resolution, R,, which is a function of the efficiency, is also a function of the volume of the stationary phase, V,, in the CCC column (see Figure 3 and comments). I n most cases, a n increase in flow rate generates a decrease of V, in the CCC column. The combination of these two variations and their consequences on R, is not fully understood and is under investigation in many laboratories.
Dual-mode operation An additional feature of CCC is its ability to be used in either normal- or reversed-phase elution with t h e same two - phase partition solvent system, as illustrated in Figure 4. The hypothetical sample contains five components (a,b,c,d, and e), whose affinity for the nonpolar upper
Figure 5. CCC technology developments over the past 20 years. Commercially available instruments appear in shaded boxes; prototype instruments appear in unshaded boxes. ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15,1991
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INSrRLJAUEN7ArION phase increases from a to e. The less polar components (c, d, and e) separate from each other via elution with the upper phase (normal-phase chromatography), whereas the more polar components (a and b) remain in the stationary lower phase (Panels I and 11).During upper - phase elution, components a and b slowly migrate and separate from each other in the stationary phase (Panel 111). After a sufficient amount of upper phase has passed through the CCC column, mobile phase and flow direction are reversed (Panel N), and components a and b are eluted. This feature makes CCC popular with natural products chemists for isolating the desired compounds from complex and diverse starting materials. Both polar and nonpolar compounds are certain to be retrieved in a single chromatographic run. CCC is also popular when biological products are involved, because irreversible adsorption to the stationary phase is not possible.
Modern instrumentation Figure 5 summarizes the main developments of CCC technology over the past 20 years. Most of the instruments have been developed and built
by Ito and co-workers (6)’and many are prototypes. Some promise outstanding utility in the near future, such as the cross-axis flow-through coil planet centrifuge (scheme X) for large- scale preparative chromatography. Some CCC instruments are now commercially available and are being used in both industry and academia. Of particular note is highspeed CCC, which has been developed primarily by Ito’s group. We will now describe two modern instruments, one of the HSES type and one of the HDES type, as well as some prototypes. C e n t r i f u g a l partition chromatograph (CPC Model LLN).According to the column’s configuration and Ito’s nomenclature, t h e CPC Model LLN device is a centrifugal droplet CCC, or centrifugal locular CCC, based on HSES. The column is made of rectangular cartridges of poly(tetrafluoroethy1ene) (PTFE) plates, engraved with channels and ducts, connected with capillaries and arranged in a circle around the rotor of a centrifuge, so that the channels are oriented parallel to the direction of the centrifbgal field (see Figure 6). Up to 12 cartridges can be used in series, thus giving 4800 channels
tary se
Figure 6. Schematic of the CPC Model LLN showing the rotor, the configuration of the cartridges, and the flow pattern in the channels that constitute the CPC column . For a cartridge type 250W, the dimensions of a channel are 12.4 x 1.1 x 2.4 mm, and there are 400 channels per cartridge. Up to 12 cartridges can be put in the rotor. The stationary phase is maintained in each channel by the centrifugal field created by the spinning rotor. The mobile phase passes through the stationary phase as small droplets or streams moving in the centrifugal field, which then form an emulsified band with the stationary phase before coalescing and being transferred to the next channel. Other types of channels exist, differing only in shape and size.
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991
with a total volume of 240 mL. The CPC column is linked to the pump and the detector through two rotary seals that support pressures up to 60 bars, and can work safely for a long period of time if carefully maintained. Two modes of elution are defined: elution with the upper phase, in the ascending mode (droplets of light phase go contrary to the centrifugal field), and elution with the lower phase, in the descending mode (droplets of heavy phase fall through the light one, in the same manner as the centrifugal field). CPC columns range from a volume of -120 mL with -2400 partition channels (six cartridges type 250W) to - 900 mL with 480 partition channels (12 cartridges type 1000E, for preparative purposes). HSES prototypes. A pilot plant scale countercurrent chromatograph, based on HSES, with a column made of stacked disks engraved with 1080 channels (V,,, -7 L) and two rotary seals for connections (the CPC 1-007 [a) has been used for purification of fatty acid ethyl esters. Up to 300 g in 400 mL can be injected, and the instrument is equipped with a motor and electrical parts that have been developed to conform to anti-explosion standards. Two prototypes of toroidal coil centrifuges (i.e., HSES systems where the column is a simple coil wound around the periphery of the rotor of a centrifuge) have been described. The TCC (toroidal coil centrifuge 181)uses a rotating seal-free arrangement (nonplanetary scheme J), with columns varying from 0.1 to 0.55 mm i.d. and 4000-10 000 turns; the rotation speed is 1000 rpm. The centrifugal countercurrent partition chromatograph (CCPC [91) uses a rotating face seal as the inlet and outlet, with one center hole and one offset hole in the rotating part and one center hole and a small circular groove on the surface of the stationary part. The columns are made of PTFE tubing of 0.6-mm i.d. The TCC column is 10 m long (710 turns, V, = 2.8 mL), and the CCPC column is 47 m long (4000 turns, Vtot = 12 mL); the rotor speed ranges from 300 to 700 rpm. For these prototypes, the injection range is 5-20 pL of -10 pg/pL solute. HSCCC. According to Ito’s nomenclature, HSCCC is a scheme J synchronous coil planet centrifuge, based on HDES. All HSCCC a p p a r a t u s share a common basic design that produces the type J synchronous planetary
1 lCjlShort
coupling pipe
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Figure 7. High-speedcountercurrent chromatograph. (a) Schematic. (b) Successive positions of the spiral column during complete revolution around the central axis of the centrifuge. The mixing zone is always located near the center of the centrifuge while the spiral column rotates around its own axis and thus travels in the column at a rate equal to the speed of revolution o. The uncoiled columns at the bottom of the diagram numbered I-IV correspond to the column position in the top diagram and show movement of the mixing zone through the spiral column. For o = 800 rpm, the two solvent phases at any portion of the column are subjected to a typical partition process of repetitive mixing and settling at a rate exceeding 13 times per second while the mobile phase is steadily passing through the stationary phase. (Adapted from Reference 6, pp. 337 and 348 by courtesy of Marcel Dekker, Inc.)
motion of the column holder; the design principle is illustrated in Figure 7a, where the orientation of the centrifuge axis is placed in the horizontal position. A large cylindrical coil holder coaxially holds a planetary gear that is coupled to an identical stationary gear (shaded) mounted on the central axis of the centrifuge. This gear arrangement produces a synchronous planetary motion of the coil holder: The holder revolves around the central axis of the apparatus and simultaneously rotates about its own axis a t the same angular velocity and in the same direction. This synchronous planetary motion of t h e holder prevents t h e twisting of the flow tubes linking the multilayer coiled column to the pump and the detector. Typically, the coiled column is a 130-m-long, 1.6-mm-i.d. PTFE tube with a total capacity of about 290 mL. Many other coil sizes (i.e., internal diameter and length of tubing, ratio of the radius for the revolution to the radius of the coil, p = r/R) can be used for analytical or preparative purposes. Two modes of elution-head-totail and tail - to - head-are defined and named (in reference to handedness of the coil and column rotation, but irrelevant to column revolution; see Figure 7b). The mobile phase that will be used in tail-to-head elu-
tion will be the upper phase if the solvent system is characterized by high interfacial tension and a hydrophobic organic phase (systems such as hexanelwat er, ethy1 acet ate/water, and chloroform/water). It will be the lower phase if the solvent system is characterized by low interfacial tension and a very hydrophilic organic phase (such as l-butanoYacetic a c i d water and 1-methylpropanol/water). HSCCC devices are very popular because they are low cost and easy to operate, and they allow 100-mgscale purification within a few hours or, sometimes, minutes. HSCCC prototypes. Three prototypes of analytical synchronous scheme J countercurrent chromatographs have been tested. They can be operated at a maximum speed of 4000 rpm (and thus they are named HSCCC -4000), and have multilayer coil columns prepared by winding an - 15-m length of a 0.85-mm-i.d., 0.55-mm-i.d., or 0.4-mm-i.d. PTFE tubing, with total capacities of about 8, 5, and 1.5 mL, respectively (8).Injection range for these prototype instruments is 5-20 pL of -10 pg/pL solute. A prototype of a preparative countercurrent chromatograph has been described, based on scheme J, with eight multilayer coils in series (the multicoil countercurrent chromato graph). The column is made of 1.6-
mm-i.d. PTFE tubing, giving a total volume of 385 mL (IO).A sample of 200 to 300 mg in 5 to 10 mL can be injected. Another preparative countercur rent chromatograph h a s been described based on a synchronous scheme X rotary seal-free arrangement (the cross-axis synchronous flow - through CPC, for cross - axis synchronous flow-through coil planet centrifuge [ I l l ) . The coiled column is 2.6-mm-i.d. PTFE tubing, 75 m long, with a total volume of 400 mL. The sample size is 4 - 40 mL. Solvent systems for CCC Selecting a two - phase solvent system for CCC is similar to choosing a column and an eluant for HPLC. Important criteria are the polarity of the sample, its solubility, charge state, and ability to form complexes. The purpose of solvent optimization for CCC separations is to find a solvent combination for which the partition coefficients of the species to be separated are different from each other. A tremendous variety of two - phase systems can be found in the literature; most of them consist of a mixture of three solvents, and some have four or more solvents. Most often with CCC, optimization of a separation involves optimization of chromatographic selectivity, and it is here that CCC has the most to of-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991 * 575 A
fer. Both phases are directly accessible, and their compositions can be fine-tuned to achieve the desired resolution. Phase diagrams can be used, when available, to intelligently understand the effects of varying the composition of one phase upon the composition of the other. Ternary diagrams for many solvent systems have been compiled by S ~ r e n s e nand Arlt (12).Such systems often consist of two immiscible solvents plus a third solvent that is soluble in the two primary solvents. For example, chloroform and water exhibit very different solvent proper ties for most substances. In many cases, solutes will strongly favor (i.e., be soluble in) either the chloroform or the water, but not both. No partitioning will occur and there will be little or no resolution of solute components. Addition of an alcohol that is soluble in both phases, such as methanol or ethanol, will cause some water to dissolve in the chloroform phase and some chloroform to dissolve in the aqueous phase, thereby bringing the solvent properties of the two phases closer together. Solutes will then be partitioned between the two phases, and fractionation of the mixed solutes will be realized. For the fractionation of water-soluble materials, consider the following solvent systems: butyl alcohols/water (aqueous salt solutions or buffers), butyl alcohols/methanol or ethanol/ water, butyl alcohols/formic or acetic acid/water, propyl alcoholdaqueous ammonium sulfate solutions, and ethyl acetate/water (aqueous salt solutions or buffers). For the fractionation of materials sparingly soluble in water but soluble in alcohols, use solvent systems such as chloroform/methanol/water, chloroform/methanol/propyl or butyl alcohols/water, chloroform/formic or acetic acid/methanol/water, and butyl alcohols/water (aqueous salt solutions or buffers). Finally, for fractionation of materia l s soluble i n hydrocarbons and ether, hydrocarbons/acetonitrile, hydrocarbons/methanol (5% water), and hydrocarbons/ethanol (10-20% water) solvent systems can be used. Applications and examples The box a t right lists some examples of applications of CCC. Many are found in the field of natural products chemistry, where CCC has been most popular. CCC is a powerful technique that is complementary to other exist ing isolation methods. Compounds with unknown properties that could 576 A
Examples of applications of CCC Separated compounds
Solvent systems
Insecticides
n-Pentane/ethanol/water
Herbicides
n-Hexane/ethyl acetate/methanol/water
Plant hormones
n-Hexane/ethyl acetate/methanol/water Chloroform/acetic acid/water
Antibiotics (siderochelineA, efrotomycin, pentalenolactone, tirandamycin A and B, valinomycin, piericidin A1 , concanamycin C, actinomycins,quinomycin A and C, tomaymycin, acivicin macquarimicin, arizonins, tiacumicins, tetracycline, nystatin, erythromycin)
Chloroform/methanol/water Carbon tetrachloride/chloroform/methanol/water
n-Hexane/methylenechloridelmethanoVwater n-Hexanelethyl acetate/methanol/water n-Hexane/methanol/water n-Hexane/diethyl ether/methanol/water Chloroform/methanol/0.2 M acetic acid
Steroids
Methylene chlorideln-hexanelmethanoVwater Methylene chloride/methanol/water
Pigments
Chloroform/acetic acid/water Ethyl acetateil -butanol/water
Tannins
1 -Butanol/O.l M NaCl 1-Butanol/l-pro anol/water Chloroform/metkanol/water
Peptides (dipeptides, gramicidins, cholecystokinin fragment, bombesin, bovine insulin, synthetic peptides)
1-8utanoVacetic acid/water Chloroform/acetic acid/formic acid/water C hloroform/benzene/methanol/water n-Hexane/l-butanol/O.l-O.5%TFA 1-Butanol/0.2-0.5M ammonium acetate
Antitumor drugs
Chloroform/dichloroethane/n-hexane/methanol/
water
Dichloroethane/methanoVbuff er n-Hexanelethyl acetate/nitromethane/methanol
Medicinal herbs Saponins
Ethyl acetate/ethanoV0.07M NaOH Chloroform/buffer Chloroform/benzene/ethyl acetate/methanol/water
C hloroform/methanol/water C hloroform/methanol/l-propanoVwater
Sugars
1-ButanoI/ethanol/water
Flavonoid glycosides, isoflavone glycosides, anthraquinone lycosides, xanthones, carjenolides, terpenoids
Chloroform/methanol/water
Alkaloids
Chloroform/methanol/water or 5% HCI
Essential oils, fatty acids
Nonaqueous solvent systems: Hexanelethyl acetate/nitromethane/methanol
Chloroform/methanol/l-propanol/water 1-ButanoVacetone/water
Heptane/acetone/methanol
Hexanehethylene chloride/acetonitrile Heptane/acetic acidhethanol n-Hexane/acetonitrile Cells, organelles, proteins
Aqueous two-phase solvent system, using two immiscible aqueous solutions of polymers such as pol ethylene glycol), dextran, Aquaphase JbT,or Reppal PES
chemically interact with a solid phase and thereby be irreversibly retained or even decomposed can easily
ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991
be handled and purified in the mild environment of CCC. The ability to inject crude extracts can drastically
Figure 8. Countercurrent chromatogram of flavonoids from a crude sea buckthorn ethanol extract. Instrument: HSCCC-4000, consisting of a coil planet centrifuge with 2.5-cm revolution radius; the column was a multilayer coil, 0.85-mm i.d. and 8 mL capacity with p = 0.51 - 0.77 (p = r / R ). Solvent system: chloroform/methanol/water (4:3:2, v/v/v), with the lower phase (chloroform rich) as mobile phase. Flow rate: 2 mL/min; rotation rate: 3500 rpm. Sample size: 120 pg. Peak 1: isorhamnetin; Peak 2: quercetin. (Adapted witk permission from Reference 14.)
reduce the initial purification steps and save time when solute stability is a problem. This may be what en-
couraged scientists to investigate possibilities of liquid-liquid partitioning as a tool for isolation of unknown but active natural products from complex and diverse starting materials. Another interesting application is in the trace enrichment of low-level components. CCC can be used as a continuous extractor, with an organic phase as the stationary phase, by percolating the dilute solution of interest (after adding salts and saturating with the organic solvent to reach equilibrium) through the stationary phase. This has been done for the extraction of urinary drug metabolites (13),and it can be done for trace enrichment of rare earth metal ions by adding a complexing agent (such as a crown ether or a phosphonic ester) to the organic phase. Figure 8 is an example of a fast and efficient separation of flavonoids from a crude ethanol extract of sea buckthorn (Hippophae rhamnoides), using an HSCCC-4000 and a twophase solvent system containing chloroform/methanol/water (4:3:2, v/v/v) (14). From the two last peaks we can roughly estimate the number of theoretical plates to be i n the range of 200-300, for a coiled column 15 m long, 0.85 mm i.d., and -140 t u r n s (i.e.) 1.5 to 2.3 theoretical plates per turn, which is very good). Figure 9 is a n example of peptide
purification (15) with a n HSCCC prototype. An additional heating system allows the chromatography to be conducted at 40-45 "C to decrease the viscosity of l-butanol, which is one of the solvent system components. Solvent systems such as l - b u tanoywater with various amounts of carboxylic acids such as dichloroacetic acid, trifluoroacetic acid (TFA), or acetic acid are very useful for the partitioning of peptides of various hydrophobicities and molecular sizes. Systems such as chloroform/acetic acidwater, or hexane/l-butanoU0.1% TFA, are used for the partitioning of more hydrophobic peptides, whereas salts (e.g., ammonium acetate) may be added for purification of more hydrophilic peptides. Figure 10 is a n example of largescale p u r i f i c a t i o n of dihomo-ylinolenic acid ethyl ester with an industrial centrifugal partition chromatograph (CPC 1-007). As already described, the capacity of the column is 7 L for 1080 channels; 130 g of a mixture of fatty acid ethyl esters is injected in a single chromatographic run. The two major peaks do not correspond to pure compounds, and thus it is difficult to estimate the efficiency of this apparatus. From the first major peak we can roughly estimate the number of theoretical plates to be - 220 (i.e., 0.2 theoretical plates per channel). The shape of the peaks
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Figure 9. Purification of synthetic bombesin (a tetradecapeptide) by CCC. (a) HSCCC of bombesin. Instrument: multilayer coil planet centrifuge, similar to the HSCCC, with an additional heating system. Solvent system: 1YOdichloroacetic acid/l-butanol (l:l, v/v), with the lower aqueous phase as mobile phase. Flow rate: 150 mUh; rotation rate: 800 rpm. (b) Analytical HPLC of 7.5 pg of crude synthetic peptide on a p-Bondapak C,8 column and a 0.1% phosphoric acid and acetonitrile gradient. (c) Analytical HPLC of 10 pg of fraction 55 of the HSCCC run. The recovery of purified peptide was - 82% by weight and more than 98% pure as determined by analytical HPLC. (Adapted from Reference 15, pp. 604 - 606 by courtesy of Marcel Dekker, Inc.)
ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15,1991
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INS7RLJMENTATlON
Peak 1
A
Peak 2
Figure 10. Purification of dihomo-y-linolenicacid ethyl ester by preparative-scale CCC with a centrifugal partition chromatograph. The column consisted of stacked disks engraved with 1080 channels with a total volume of -7 L. The solvent system was hexane/acetonitrilewith the lower phase (acetonitrile) as the mobile phase. Flow rate: 150 mumin; rotational speed: 700 rpm. Sample: 130 g of a mixture containing 17.08% of dihomo-y-linolenic acid ethyl ester, dissolved in 300 mL of upper phase + 100 mL of lower phase; injection at 20 mumin. Detection: UV at 210 nm; cell length: 0.2 mm, with the range dial to 2.0 AUFS (full scale of the recorder is 100 AUFS). Peak 1: mixture of linolenic acid ethyl ester (C,8:3) and arachidonic acid ethyl ester (C20:4); Peak 2: mixture of linoleic acid ethyl ester (C,8:2) and dihomo-y -linolenic acid ethyl ester (C20:3). The second peak was analyzed by GC, and percentages are indicated.
does not indicate overloading of the column for this injection (130 g), and we can consider larger injection without significant loss in efficiency. Just as with HPLC, using gradient elution in CCC provides an easy way to fractionate solutes of widely differing polarities and partition coefficients as well as to reduce run times. The easiest way to design a gradient
for CCC is to refer to ternary diagrams corresponding to the ternary liquid mixtures widely used to build up two-phase systems. Figure 11 is an example of gradient elution with the system hexane/ 1-butanovwater (+ 0.5% TFA); the water-rich phase is the stationary phase (16). Compositions of initial (IMP) and final (FMP) mobile phas-
es, as well as stationary (SP)phase are calculated from the phase diagram. Elution is reversed after the gradient (the water-rich phase becomes the mobile phase) to yield the hydrophilic tripeptide Tyr - Gly - Gly. One benefit of the gradient approach is that solvent composition is optimized for each component of a complex mixture. Because one can use octanol and water (mutually saturated) as a solvent system, CCC offers a unique and powerful alternative to the classic shaking flask method for determination of the octanol-water partition coefficient, Kow. The general equation is directly accessible
where VR is the retention volume and Vw and Vo are the volumes of water and octanol, respectively, in the CCC column; t h e water is t h e mobile phase. Octanol can be used as the mobile phase instead of water, and the CCC apparatus can be filled with a controlled ratio of the two phases to expand the range for the KO, determination (17).Furthermore, dual-mode elution (see Figure 4 and comments) can be applied as follows. Water is first used as the mobile phase, and a lipophilic analyte that will travel very slowly and stay in the instrum e n t is injected. After a l a r g e a m o u n t of w a t e r , VI, h a s been pumped through, the elution mode is reversed, the octanol becomes the mobile phase, and the analyte goes back with a retention volume in the reverse mode V,. The KO, is then independent of every parameter except
Figure 11. Gradient elution of a complex peptide mixture with the hexane/l -butanol/water + 0.5% TFA system. (a) Composition of initial (IMP) and final (FMP) mobile phases and of the stationary (SP) phase are calculated from the phase diagram. (b) Chromatogram of the mixture. Instrument: Centrifugal partition chromatograph, CPC Model LLN. Mobile phase: hexane/butanol-richphase, ascending mode. Elution reversed at t = -1 35 min. Flow rate: 4 mumin; Rotation speed: 700-800 rpm. Pressure drop: 47-40 bars, room temperature. Detection: UV at 280 nm, 0.7 AUFS. Sample in 4 mL.
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Vl and V2 so that Kow = VI/& (5) Can it be more simple? This method complements the normal elution mode for determining Kow, with a demonstrated range of 75 - 3091 (181, which has been recently extended from 29 to 12 300 (1.5 to 4.5 log Kow) (19).
Perspectives A study of t h e recent literature shows a steady growth in the number of CCC publications since 1970. Especially noteworthy is the contribution Ito has made to the field. During the past 20 years, he has co-authored 38% of all CCC papers published. As mentioned previously, most of the publications deal with natural products chemistry and instrumentation. Purification of peptides is a n emerging but very promising area where CCC can successfully replace standard, expensive, reversed - phase preparative-scale HPLC. The use of aqueous two-phase solvent systems-for example, systems made of two immiscible aqueous polymer phases such as solutions of dextran and poly(ethy1ene glycol)-to separate blood cells, particles, organelles, biopolymers, and biomembranes by CCC is a matter of active research. As the literature suggests, the popularity of CCC has been steadily increasing. It is now a standard when natural products or labile molecules are involved and can be used in areas such as peptide purifications, metal ion enrichment and extraction, and chiral separations. As these fields advance, they will contribute to a better understanding and acceptance of this original chromatography. Although it has achieved popularity in some research communities, CCC must evolve in two directions to attract a large number of users from industry and research. First, efficiency must be increased so that separation can be achieved more quickly for making the method more competitive with HPLC for complex separations. Modern CCC works at the hour and 1000-plate scale; to reach the minute and 10 000-plate scale will require much research. Second, capacity must be increased, and this is where CCC could become especially attractive. Because of the very low cost of the packing (i.e., solvents), costs for preparative CCC are essentially capital equipment costs rather than consumable operating costs. It is easy and fast to refresh a CCC column after a run simply by flushing the CCC apparatus with a fresh mix-
ture of stationary and mobile phase. This is inexpensive when compared with the cost of HPLC packing material.
References (1) Ito, Y.; Weinstein, M.; Aoki, I.; Hara-
da, R.; Kimura, E.; Nunogaki, K. Nature
1966,212,985. (2) Ito, Y.; Conway, W. D. Anal. Chem. 1984,56,534 A. (3) Rosset, R.; Caude, M.; Jardy, A. In
Manuel Pratique de Chromatographies en Phases Liquide et en Phase Supercritique; Masson: Paris, in press. (4) Armstrong, D. W.; Bertrand, G. L.; Berthod, A. Anal. Chem. 1988, 60,2513. (5) Ito, Y.; Oka, H., Laborato of Biophysical chemistry, National Ynstitutes of Health, personal communication,
1991. (6) Ito, Y. In Countercurrent Chromatogra-
phy: Theory and Practice; Mandava, N. B.; Ito, Y., Eds.; Dekker: New York, 1988; p. 79 and references therein. (7) Kosuge, Y.; Nakaya, N.; Bando, Y.; Muramaya, W. Presented at the Third International Colloquium on Centrifugal Partition Chromatography, San Mat e ~CA, , June 1990. (8) Oka, H.; Ikai, Y.; Kawamura, N.; Yamada, M.; Harada, K.; Suzuki, M.; Chou, F-E.; Lee, Y-W.; Ito, Y. J. Liq. Chromatogr. 1990, 13, 2309. (9) Kim, L-S.; Tian, Z-Q.; Li, X-N.; Zhang, Y.; W a g , J.J Chnnnutogx 1989,483,359. (10) Knight, M.; Gluch, S. J. Liq. Chromatogr. 1990, 13, 2351. (11) Ito, Y. Sep. Sci. Technol. 1987,22,1971 and 1989. (12) Starensen, J. M.; Arlt, W. Liquid-Lquid Equilibrium Data Collection;Dechema: Frankfurmain, 1980; Vol. V, Parts 2 and 3. (13) Nakazawa, H.; Riggs, Jr., C. E.; Egorin, M. J.; Redwood, S. M.; Bachur, N. R.; Bhatnagar, R.; Ito, Y. J. Chromatogr. 1984,307,323.
(14) Oka, H.; Oka, F.; Ito, Y. J. Chromutogr. 1989, 479, 53. (15) G i g h t , M. In Countemrrent Chroma-
tography: Theory and Practice; Mandava, N. B.; Ito, Y., Eds.; Dekker: New York, 1988, p. 583 and references therein. (16) Foucault, A.; Nakanishi, K. J. L q . Chromatogr. 1990, 13, 3583. (17) Vallat, P.; El Tayar, N.; Testa, B.; Slacanin, I.; Marston, A.; Hostettmann, K. J. Chromatogr. 1990,504,411. (18) Menges, R. A.; Bertrand, G. L.; Armstrong, D. W. J. Liq. Chromatogr. 1990,
13, 3061. (19) Gluck, S. J.; Martin, E. J.J. L q . Chromatogr. 1990, 13, 3559.
Suggested reading Countercurrent Chromatography: 73eory and Practice; Mandava, N. B.; Ito, Y., Eds.; Dekker: New York, 1988. Countercurrent Chromatography, Apparatus, Theory & Practice; Conway, W. D., Ed.; VCH Publishers: New York, 1990. Ito, Y. J. Biochem. Biophys. 1981, 5, 105. Mandava, N. B.; Ito, Y.; Conway, W.D. Am. Lab. 1982, 14(10), 62. Mandava, N. B.; Ito, Y.; Conway, W. D. Am. Lab. 1982,14(11), 48. Ito, Y. CRC Crit. Rev. Anal. Chem. 1986, 17, 65.
Foucault, A,; Rosset, R. Analusis 1988, 16(3), 157.
Mandava, N. B. J. Liq. Chromatogr. 1984, 7(2), 227-432 (special issue on CCC edited by Mandava). Mandava, N. B. J. fiq. Chromatogr. 198S, 8(12), 2127-2336 (special issue on CCC edited by Mandava). Cazes, J. J. Liq. Chromatogr. 1988, 11(12), 2433-2546 (special issue on centrifu a1 partition chromatography edited f y Cazes). Mandava, N. B. J. Liq. Chromutogr. 1990, 13(12), 2307-2512 (special issue on CCC edited by Mandava). Cazes, J.J. Liq. Chromatogr. 1990, 13181, 3559-3710 (special issue on centrifu a1 partition chromatography edited %y Cazes).
W
u
Alain €? Foucault received a degree in engineeringfrom the Ecole Supkrieure de Physique et de Chimie Industrielles de Paris (France) in 1969 and a Ph.D. in analytical chemistty from the Universiti Pierre et Marie Curie (Paris, France) in 1975. His research interests, which initiallyfocused on ligand-exchange chromatography, expanded to include CCC and led to a three-year collaboration with K. Nakanishi at Columbia University. He is pursuing CCC work with F. Le Gofic at the Ecole Nationale Supirieure de Chimie de Paris. ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15,1991
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