Countercurrent Chromatography
Alain P. Foucault Laboratoire de Bioorganique et Biotechnologies Ecole Nationale Supérieure 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 t h e pioneering studies of Ito, Nunogaki, and co-workers in J a p a n (1). 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 t h e way to the fruitful research conducted by Ito in the United States, which h a s been devoted primarily to the study 0003-2700/91/0363-569A/$02.50/0 © 1991 American Chemical Society
INSTRUMENTATION 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 a n d 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. C C C and HPLC
CCC and HPLC are similar in several respects. Both are based on t h e same fundamental mechanism, t h e partitioning of solutes, which determines the distribution of the molecules in t h e various phases. Both techniques have t h e 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 become 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 a n d HPLC s h a r e t h e same technologies. The same pump, injector, and detector can be used, although minor restrictions can arise in certain cases. Figure 1 is a schematic comparison between HPLC and CCC. For convenience, we have chosen a reversedphase chromatographic process. In HPLC (left), the stationary phase is an organic moiety bonded to silica and solvated mainly by the organic solvent of the aqueous organic mobile phase. The volume ratio of the sta-
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INSTRUMENTATION
(c) Reversed-phase CCC
(a) Reversed-phase HPLC Silica
Hexane (stationary phase)
Hexyl bonded to silica (stationary phase)
Water + methanol (mobile phase)
Water + methanol (mobile phase)
vm + v,.vM
Vn+Vs-Vw-Vsi
m Porous + interstitial volumes (-75%) *
Impregnated liquid stationary phase (-5%)
Liquid stationary phase
Silica skeleton (-20%)
Mobile X phase
Volume
Ratio depends on the experimental conditions (-50-80%)
Volume
Packed column liquidliquid chromatography
CCC
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/methanol to flow through the hexane (saturated primarily with methanol) stationary phase, (d) The total volume is used for the partition process, and the ratio Vs/ Vm, depending upon the experiment, is - 50:50. Abbreviations: Vs, Vm, Vtot, and Vsi are, respectively, the volumes of stationary and mobile phases, total volume, and volume of solid silica gel.
gravitational field (G + œ2r) varies with the position of the analyzed point in the coil (basically a rotating coil t h a t exhibits an "Archimedean screw" effect). For both cases, the constant gravitational field can be augmented with a revolutionary rotation around an extra axis (R is 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 t h a t 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 p a r t i t i o n 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. H D E S 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). I m p r o v e m e n t s on t h e o r i g i n a l technology have provided fast and efficient instruments. Although not yet comparable in speed and efficiency to HPLC, the CCC devices compensate
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. In CCC (right), the silica is r e placed with a strong gravitational field that "frees" 20-30% of the total volume. (The porosity factor, e c , 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 an immiscible organic solvent (e.g., hexane) as the stationary phase; the heavier droplets of the water-organic mixt u r e pass t h r o u g h the s t a t i o n a r y phase. The volume ratio of the stationary phase is 5 0 - 8 0 % for most cases; the actual value depends on the i n s t r u m e n t , the experimental conditions, and the composition of the ternary mixture used to prepare the mobile and stationary phases.
ized as a stationary coiled tube (r is the radius of the coil) subjected to a gravitational field, G, which is constant at 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 subject to a constant gravitational field, G . The resulting
CCC: basic 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 ω is the speed of revolution.
Upper phase
Revolution
Lower phase
Flow
Mild mixing
Transfer
[Revolution Rotation Flow Mild mixing Vigorous and transfer mixing
Figure 2. Two basic CCC systems.
Ito defined two basic systems of CCC (Figure 2). The first is called the hyd r o s t a t i c e q u i l i b r i u m s y s t e m , or HSES. The "column" can be visual-
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INSTRUMENTATION for these shortcomings with attrac tive features such as the large capac ity of the stationary phase, total re covery of a sample, and low cost of the "packing" (i.e., solvents).
Less polar phase More polar phase
CCC column
CCC: advantages An advantage of CCC is that the vol ume 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 an HPLC column for the same total volume. Overload ing is uncommon in CCC; the limit ing factor is the solubility of the sam ple in both the stationary and mobile phases rather than the amount in jected. This makes CCC very attrac tive for preparative-scale chroma t o g r a p h y , w h e r e i t is a l r e a d y competitive and frequently b e t t e r t h a n preparative-scale HPLC. For the same column volume, one can pu rify larger quantities with CCC with total recovery (no adsorption) and low operating costs (the cost of the solvents only).
Normal-phase elution
Recycle
Reversed-phase elution
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 ad jacent peaks is Rs = (1/4) (a-1) NV2 [k i/(k ί + 1)]
(1)
where a is the separation factor («27 «Ρ, «ί is the capacity factor for the peak 1, and Ν is the number of theo retical plates. The term k ί is related to the partition coefficient Kx by
K = UVJVJ
Figure 3. Relationship for two ad jacent peaks between the resolution Rs, the number of theoretical plates N, and the percent of stationary phase %VS in a chromatographic column for HPLC and CCC. (a) For a given W, resolution is better in CCC than in HPLC. (b) CCC requires fewer theoretical plates than HPLC to provide the same resolution. The partition coefficients ΚΊ, K2, and the separation factor α are assumed to be constant. The volume percent of the stationary phase is - 5 % for HPLC and is usually 50 - 80% for CCC.
(2)
where Vs and Vm are the stationary and mobile-phase volumes, respec tively. Kx 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 mo bile phase. Using these relationships, we can replace k [ in Equation 1 and obtain RB = (1/4) (ct-1) Ν1'2 χ « ζ / [Kt + (VJVS)]}
Efficiency and resolution (3)
The term V,JVe, which is always large in HPLC ( - 1 3 - 2 0 ) , is much
572 A · ANALYTICAL CHEMISTRY, VOL. 63, NO. 10, MAY 15, 1991
smaller in CCC (0.25-1 for a station ary phase occupying 50-80% 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 3 a). Put another way, it means that the number of plates required to ob tain a given resolution is less impor tant in CCC than in HPLC (Figure 3b). As shown in the example in Figure 3, for α = 1.2 and Kx = 1, 1000 theoaretical plates will give Rs = 0.1 for HPLC (Vs - 7% of the total volume), and Ra = 1 for CCC (Va - 65% of the total volume). To obtain Rs = 1.5, -185 000 plates will be required in HPLC, whereas 2200 plates will be enough for CCC (Figure 3b).
If we can minimize t h e 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
Hydrostatic equilibrium system (constant gravitational field) Earth gravity
Locular CCC
Rotation locular CCC
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, Ra, which is a function of the efficiency, is also a function of the volume of the stationary phase, Vs, in the CCC column (see Figure 3 and comments). In most cases, an increase in flow rate generates a decrease of F s in the CCC column. The combination of these two variations and their consequences on RB is not fully understood and is under investigation in many laboratories.
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 an instrument based on HSES (the CPC Model LLN) to show that van Deemtertype plots exhibit m a x i m a r a t h e r t h a n minima. Consequently, the best efficiency is obtained at high or low flow rates. This observation is significant because analogous flow rates a r e often u s e d for CCC a n d for HPLC; unfortunately, this leads to the worst efficiency. It is far better to
Droplet CCC
Gyration locular CCC
Dual-mode operation An additional feature of CCC is its ability to be used in either normal- or reversed-phase elution with the same t w o - p h a s e partition solvent system, as illustrated in Figure 4. The hypothetical sample contains five c o m p o n e n t s (a,b,c,d, a n d e), whose affinity for the nonpolar upper
Hydrodynamic equilibrium system (variable gravitational field)
Centrifugal field
Earth gravity
Rotary coil assembly (Type J, planetary and nonplanetary)
Centrifugal field Angle rotor coil planet centrifuge (Type I - L)
Flow-through coil planet centrifuge (Type I)
Horizontal CCC
Type J planetary gear drive
Toroidal coil planet centrifuge
Centrifugal partition chromatograph One rotation axis Column is locular Two rotary seals Medium back pressure mg to hundred g scale
Nonsynchronous flow-through coil planet centrifuge (Type I)
Elution centrifuge (Type L)
Countercurrent extraction coil planet centrifuge
High-speed CCC Toroidal coil centrifuge (Type J, nonplanetary)
Combined flow-through coil planet centrifuge (Types I and J)
Two gyration axis Column is long Teflon tubing No rotary seal Low back pressure mg to g scale injection
Cross-axis flow-through coil planet centrifuge (Type X) Potential 10-20 g injection range
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 · 573 A
Figure 7. High-speed countercurrent 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 locat ed 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 ω. The uncoiled columns at the bottom of the diagram numbered l-IV correspond to the column position in the top diagram and show movement of the mixing zone through the spiral column. For ω = 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 de sign principle is illustrated in Figure 7a, where the orientation of the cen trifuge axis is placed in the horizon tal 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 h o l d e r : The h o l d e r revolves around the central axis of the appa r a t u s and simultaneously r o t a t e s about its own axis at the same angu lar velocity and in the same direc tion. This synchronous planetary mo tion of t h e h o l d e r p r e v e n t s 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., inter nal diameter and length of tubing, ratio of the radius for the revolution to the radius of the coil, β = rlR) can be used for analytical or preparative purposes. Two modes of elution—head-totail and t a i l - t o - h e a d — a r e defined and named (in reference to handed ness 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 hydro phobic organic phase (systems such as hexane/water, ethyl acetate/water, and chloroform/water). It will be the lower phase if the solvent system is characterized by low interfacial ten sion and a very hydrophilic organic phase (such as 1-butanol/acetic acid/ 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 proto t y p e s of a n a l y t i c a l s y n c h r o n o u s scheme J countercurrent chromatographs have been tested. They can be operated at a m a x i m u m 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). In jection range for these prototype in struments is 5-20 μϋι of -10 μg/μL solute. A prototype of a preparative coun tercurrent 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 (10). A sample of 200 to 300 mg in 5 to 10 mL can be injected. Another preparative countercur rent chromatograph h a s been de scribed b a s e d on a s y n c h r o n o u s scheme X rotary seal-free arrange m e n t (the cross-axis synchronous flow-through CPC, for cross-axis synchronous flow-through coil planet centrifuge [11]). 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 col umn and an eluant for HPLC. Impor tant 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 sepa rated are different from each other. A t r e m e n d o u s v a r i e t y of t w o - p h a s e systems can be found in the litera ture; most of them consist of a mix ture 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-
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INSTRUMENTATION 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 S0rensen and 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 properties 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 t h a t 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 alcohols/aqueous a m m o n i u m 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 materials soluble in h y d r o c a r b o n s a n d ether, hydrocarbons/acetonitrile, hyd r o c a r b o n s / m e t h a n o l (5% w a t e r ) , and hydrocarbons/ethanol (10-20% water) solvent systems can be used.
Applications and examples The box at 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 existing isolation methods. Compounds with unknown properties t h a t could
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 (siderocheline A, 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/methylene chloride/methanol/water n-Hexane/ethyl acetate/methanol/water n-Hexane/methanol/water n-Hexane/diethyl ether/methanol/water Chloroform/methanol/0.2 M acetic acid
Steroids
Methylene chloride/n-hexane/methanol/water Methylene chloride/methanol/water
Pigments
Chloroform/acetic acid/water Ethyl acetate/1 -butanol/water
Tannins
1-Butanol/0.1 M NaCI 1 -Butanol/1 -propanol/water Chloroform/methanol/water
Peptides (dipeptides, gramicidins, cholecystokinin fragment, bombesin, bovine insulin, synthetic peptides)
1 -Butanol/acetic acid/water Chloroform/acetic acid/formic acid/water Chloroform/benzene/methanol/water n-Hexane/1-butanol/0.1-0.5% TFA 1 -Butanol/0.2-0.5 M ammonium acetate
Antitumor drugs
Chloroform/dichloroethane/n-hexane/methanol/ water Dichloroethane/methanol/buffer n-Hexane/ethyl acetate/nitromethane/methanol
Medicinal herbs
Ethyl acetate/ethanol/0.07 M NaOH Chloroform/buffer
Saponins
Chloroform/benzene/ethyl acetate/methanol/water Chloroform/methanol/water Chloroform/methanol/1 -propanol/water
Sugars
1 -Butanol/ethanol/water
Flavonoid glycosides, isoflavone glycosides, anthraquinone glycosides, xanthones, cardenolides, terpenoids
Chloroform/methanol/water Chloroform/methanol/1-propanol/water 1 -Butanol/acetone/water
Alkaloids
Chloroform/methanol/water or 5% HCI
Essential oils, fatty acids
Nonaqueous solvent systems: Hexane/ethyl acetate/nitromethane/methanol Heptane/acetone/methanol Hexane/methylene chloride/acetonitrile Heptane/acetic acid/methanol n-Hexane/acetonitrile
Cells, organelles, proteins
Aqueous two-phase solvent system, using two immiscible aqueous solutions of polymers such as polyjethylene glycol), dextran, Aquaphase PPT, or Reppal PES
chemically interact with a solid phase and thereby be irreversibly retained or even decomposed can easily
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be handled and purified in the mild environment of CCC. The ability to inject crude extracts can drastically
Isorhamnetin
Quercetin
Time (min) 0
4
8
Time (min) 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 β = 0.51 - 0.77 (β = ri R ). Sol vent 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 μg. Peak 1 : isorhamnetin; Peak 2: quercetin. (Adapted with 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 p a r t i tioning as a tool for isolation of un known 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 organ ic phase as the stationary phase, by percolating the dilute solution of in terest (after adding salts and satu r a t i n g with the organic solvent to reach equilibrium) through the sta tionary phase. This has been done for the extraction of urinary drug me tabolites (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 b u c k t h o r n {Hippophae rhamnoides), using an HSCCC-4000 and a twop h a s e solvent s y s t e m c o n t a i n i n g 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 in t h e 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 an example of peptide
Time (min)
purification (15) with a n HSCCC prototype. An additional heating sys tem allows the chromatography to be conducted at 4 0 - 4 5 °C to decrease the viscosity of 1-butanol, which is one of the solvent system compo nents. Solvent systems such as 1-butanol/water with various amounts of carboxylic acids such as dichloroace tic 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 acid/water, or hexane/l-butanol/0.1% 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 an example of larges c a l e p u r i f i c a t i o n of d i h o m o - γ linolenic acid ethyl ester with an in dustrial 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 cor respond to pure compounds, and thus it is difficult to estimate the efficien cy 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
Time (min)
Time (h)
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: 1% dichloroacetic acid/1-butanol (1:1, v/v), with the lower aqueous phase as mobile phase. Flow rate: 150 mL/h; rotation rate: 800 rpm. (b) Analytical HPLC of 7.5 μ
