Analytrca Chtmlca Acta, 236 (1990) 63-16 Elsevter Sctence Pubhshers B.

V , Amsterdam

63

Liquid-liquid partition chromatography in natural product isolation

K HOSTETTMANN Instrtute * and A. MARSTON

of Phytochemtstry and Pharmacognosy, School of Pharmacy, Umversrty of Luusanne, Rue Vudlermet 2,

CH-1005 Lausanne (Swrtzerland) (Received 13th November 1989)

ABSTRACT The apphcatrons of droplet countercurrent chromatography and centnfugal partrtion chromatography (CPC) to the tsolatton and punfrcatton of natural products are described.. The former IS a well established technique which has been used extenstvely m the tsolatron of polar substances and the fracttonatron of crude plant extracts The recent mtroductron of dtfferent CPC techrnques and mstruments has permrtted a reductron of separation times Two of these instruments (the cartridge centnfugal partmon chromatograph and the h&-speed cod separator-extractor) have been apphed to the separation of natural products of wtdely drffermg polanttes They offer the advantage of bemg able to reverse the solvent flow. Other developments are in progress to further Improve the capablhtres of these chromatographs A descnptron of the different mstruments IS provtded, together wtth detads of their mode of operatron and a drscusston of expenmental parameters, such as flow-rates, rotation speeds, solvent selectron and the apphcatton of aqueous and non-aqueous systems. Keywords Droplet countercurrent chromatography, Centnfugal partttron chromatography, Natural products

Liquid-liquid partition chromatography, as described here, is a separation method which does not require a sorbent. As a result, it benefits from a number of advantages over liquid chromatography: there is total recovery of the introduced sample and no irreversible adsorption on a solid matrix, tailing is minimized, denaturation of sample is minimal and solvent consumption is low. Mixtures to be separated can vary from milligram to gram amounts and crude extracts pose no problems. Consequently, the isolation of natural products is an obvious application of liquid-liquid partition chromatography. The increasing use of this technique is due to the introduction over the last 15 years of a totally new series of instruments capable of performing the separations. Largely as a result of the work of Mandava and Ito [l], a variety of different prototype chromatographs have appeared but, of these, only a few have as yet
0003-2670/90/$03 50 0 1990 - Elsevrer Science Pubhshers B.V

gained popular acceptance [2]. Three of the chromatographs are outlined here, in an attempt to explam their principles of operation, choice of experimental conditions and applications. These instruments are the mostly widely used of this new generation of all-liquid systems. The first, droplet countercurrent chromatography (DCCC) [3], is one of the earlier introduced liquid-liquid separation techniques which relies on the partition of a solute between two immiscible solvents, the relative proportions of solute passing into each of the two phases being determined by the respective partition coefficients. Retention of stationary phase relies on gravity and the technique is slow. Centrifugal partition chromatography (CPC), on the other hand, relies on a centrifugal force to retain the stationary phase, and mobile phase can consequently be pumped at much higher flow-rates. Most practical applica-

64

K HOSTETTMANN

AND A MARSTON

tions have Involved either a cartridge system or a rotating coil instrument, both of which are described below.

efficient partitioning of the solute between the two phases. Separation occurs according to the drfference in the partition coefficients of the components of the sample. Choice of solvent system The formation of droplets, which 1s necessary for DCCC, depends on several factors: the drfference in density between the two liquid phases, the viscosity of the solvents, the interfactal tension, the rate of flow of the mobile phase and the inside diameter of the columns. For successful DCCC separations the choice of the two-phase solvent system is therefore crucial. Binary systems are impractical for the formatton of suttable droplets because of the large difference in polanty between the two components, and ternary (or quaternary) systems are required, such that the addition of a thud (or fourth) component, rnisc~ble with the other components, dimmishes the difference in polarity between the two phases. The selectivity of the system is thus mcreased, allowing the separation of closely related substances. The

DROPLET

COUNTERCURRENT

CHROMATOGRAPHY

Apparatus and prmclple of operation A typical DCCC instrument consists of 200-600 vertical columns (20-60 cm in length) of narrowbore silanized glass tubing, interconnected in series by capillary Teflon tubes. The whole apparatus is first filled wrth the stationary phase of a biphasic solvent system and the sample is injected. The mobile phase is then pumped via the sample chamber into the first of the columns, forming a stream of droplets in the immiscible stationary phase. Depending on the choice of solvents for the mobile and stationary phases, these droplets are made either to ascend (ascending mode) or descend (descending mode) through the columns [4]. As the mobile phase moves through the column in the form of droplets, turbulence promotes
TABLE Aqueous Substances 1 and non-aqueous separated DCCC solvent systems

Solvent system CHCl,-MeOH-H,O (7 + 13 + 8 and other proportions) CHCl,-MeOH-acetate buffer (pH 3 6) (9 + 12 + 8) CHCl,-MeOH-]% aq. NH, (7 + 12 + 8) CHCl,-MeOH-5% HCl(5 + 5 + 3) CHCl,-MeOH5% aq CaCl,-n-PrOH (50 + 60 + 40 + 6) CHCl,-MeOH-H,O-n-PrOH (9 + 12 + 8 + 1) CHCl,-MeOH-H20-n-PrOH (5 + 6 + 4 + 1) CHCl,-MeOH-H,O-n-BuOH (10 + 10 + 6 + 1) CHC13-MeOH-H,O-n-PrOH-EtOH (9 + 6 + 8 + 1+ 8) CHCl,-MeOH-H,O-C,H,Me (5 + 7 + 2 + 5) CH,Cl,-MeOH-H,O (8 + 13 + 7) n-BuOH-HOAc-H,O (4 + 1 + 5) n-BuOH-MeOH-H,O (5 + 1+ 5) n-BuOH-n-PrOH-H,O (2 + 1+ 3) n-BuOH-EtOH-Hz0 (4 + 1+ 5) n-BuOH-MezCO-H,O (33 + 10 + 50) I-BuOH-TFA-H,O (120 + 1+ 160) L&t petroleum-EtOH-H,O-EtOAc (5 + 4 + 1 + 2) CsH,,-Et,O-n-PrOH-95% EtOH-H,O (4 + 8 + 3 + 5 + 4) GH,,-EtOAc-MeNO,-MeOH (8 + 2 + 2 + 3) C,H,,-C,H,C12-MeOH (47 + 6 + 72) C,H,,-Me&O-MeOH (5 + 1 + 4) C,H,,-CH,C12-MeCN (10 + 3 + 7)

Glycosldes, tanmns, alkaloids, antlblotlcs, etc Alkaloids Basic steroid sapomns Alkaloids Glycohplds Sapomns, mdold glycosldes, xanthone glycosldes, flavonold glycosldes, anthraqumone glycosldes Sapomns Flavonold glycosldes Sapomns Alkaloids Sapomns Anthocyamns, flavonold glycosldes, peptldes Naphthahde glycosldes Tanmns Indold and secolndold glycosldes Sennosldes Peptldes Glbberelhns, anthraqumones Dlterpenolds, coumanns Essential 011s Tnterpenes, steroids Tnterpenes, steroids, depsldes Tnterpenes, steroids

LIQUID-LIQUID

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PRODUCT

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65

third component also serves to diminish the interfacial tension and viscosity of the system. Methanol is often used as an auxiliary solvent because small changes in the amount of methanol lead to large changes in the polarity of the system. Some representative systems used for the DCCC separation of natural products are shown in Table 1. As the glass tubes and interconnecting Teflon capillaries are chemically inert, the DCCC apparatus is compatible with solvent systems containing acids, alkalis and all organic solvents. Non-aqueous systems also exist (Table 1) and these are important for the separation of weakly polar compounds, especially those which are unstable m the presence of water or those which may decompose during chromatography on silica gel

151.
As a means of selecting a suitable solvent system for DCCC separations, a thin-layer chromatographic (TLC) investigation of the mixture or extract may be made [4]. Migration of the sample on silica gel plates with the water-saturated organic layer of a two-phase aqueous solvent system is carried out and if the R, values of the compounds to be separated are higher than about 0.50, the less polar layer is suitable for use as the mobile phase. For more polar solutes (R, < 0.50), the more polar layer is used as the mobtle phase (Fig. 1). When the R, values are in the range 0.40-0.60, the

Mobile phase: less polar layer

separation can be achieved by using either the more or the less polar layer as the mobile phase. If the R, values of the compounds to be separated are either too low (R, -C0.20-0.30) or too high (R, > 0.70-0.80), no elution will take place m a reasonable time and a different solvent system should be considered. However the separation of a rmxture with components of widely ranging polarities is possible if first the organic phase is used as mobile phase to elute the less polar compounds. The polar components remainmg in the instrument are pumped out and rechromatographed, using the aqueous phase as the mobile phase for their separation. This method has been employed to effect the separation of ethyl acetate-soluble acids from the seeds of Phaseolus cocczneus (Leguminosae) [6]. For non-aqueous solvent systems, TLC on silica gel is not suitable. Instead, chemically bonded phases, e.g., RP-8, are employed [7]. Although liquid chromatographic (LC) analysis of the distribution of a sample between two immiscible phases is also possible, TLC provides a very simple and rapid prehminary indication of the apphcabihty of any given two-phase solvent combination to DCCC. An alternative method of selecting suitable solvents is to monitor the distribution of 5-10 mg of sample between 5-10 ml of each of the two phases constituting the solvent system [8]. A system in which 15-25% of the sample is distributed in one of the phases is chosen. The stationary phase employed is the one that contains the most sample. Appkations The first documented use of DCCC was in the baseline separation of an artificial mixture of seven DNP-amino acids with CHCl,-HOAc-0.1 M HCl (2 + 2 + 1) as solvent system, in the ascending mode. The resolution was relatively high (900 theoretical plates for dinitrophenylalanme) [9]. Most subsequent DCCC separations have also involved polar compounds, including glycosides. Of special note are the separations of polyphenols, tannins, anthocyanins and saponins. CHCl,-MeOH-H,O systems of varying compositions are the most widely used, in view of their good droplet-forming characteristics and convenient viscosities (Table

04 Sample

T-7
Mobile phase: more polar layer

04 Sample

Fig 1. Choice of DCCC solvent system. Support, eluent, less polar layer of two-phase solvent system

sdlca

gel;

66

K HOSTEITMANN

AND A MARSTON

1). Chlorinated solvents which have high densities and low viscosities are particularly suitable for use as descending mobile phases. There has been limited use of non-aqueous solvent systems for the separation of mono-, di- and triterpenes, steroids and iridoids. Mixtures of relatively apolar substances can also be effectively separated by water-containing solvent systems. These applications have been reviewed [2,4,5], but certain examples will be presented here to illustrate the utility of DCCC for sample preparation. Sample loadings of up to 6.4 g of crude extract on columns of 3.4 mm i.d. have been possible [lO,ll] and, exceptionally, a sample size of 16 g was achieved with 10 mm i.d. columns [12]. In this latter example, DCCC was utilized as an intermediate step in the purification of a water extract from the starfish Luldiu maculutu. Eighteen columns were employed, with water saturated with n-butanol as stationary phase and n-butanol saturated with water as mobile phase, at a flow-rate of 100 ml h-. Enzymic hydrolysis of the purified mixture of steroid oligoglycosides allowed the isolation of five aglycones. Polyphenols. The acidic hydroxyl groups in polyphenols often cause irreversible adsorption on solid stationary phases during open-column and LC procedures. DCCC provides a useful altemative, therefore, for their separation, as no sorbent is involved. Further, polyphenols often occur as polar glycosides and these are fortunately soluble in the solvent systems employed in DCCC. For these reasons, separations of flavone glycosides provide a large number of applications of the method. The pure flavonoid glycosides 6-hydroxyluteolin 7-0-glucoside and 6-hydroxyluteolin 7-0gentiobioside have been obtained from a crude methanol extract of Lomutogonium curinthiucum (Gentianaceae) with the solvent system CHCl,MeOH-n-PrOH-H,O (5 + 6 + 1 + 4) (descending mode) and 3.4 mm i.d. columns [ll]. The fourcomponent solvent system is a variant of the basic CHCl,-MeOH-H,O system. A further useful solvent system for the separation of flavonoid glycosides is CHCl,-MeOH-n-BuOH-H,O (10 + 10 + 1 + 6), employed in the descending mode to obtain the C-glycosylflavones isovitexin, iso-

OH

R
1 2 3 4 5 6 7 8 9 10 11 12 OH OH OH H OH OH OH OH OH OH H OH

R2
Glc'-Glc Gal'-Apl Gl&Apl Gl&API An-API Xyl'-API Gal Glc Al-a XYl Ara Rha

OCCC, ascending mode CHCl,-MeOH-"BuOH-H,O

LH-20 or RP-8

Fig 2. Isolation of flavonol glycosldes from Secundaca oersrfolrn (Polygalaceae)

dr-

orientin, vicenin-2 and lucenin-2 from the leaves of Lespedezu cuneutu (Leguminosae) [13]. The same biphasic system was used in the isolation of twelve flavonoid glycosides from Securiducu diverszfohu (Polygalaceae). DCCC of a methanol extract of the leaves (in the ascending mode) in conJunction with Sephadex LH-20 gel filtration and low-pressure reversed-phase chromatography was sufficient to obtain all the pure flavonoids (Fig. 2) [14]. Tannins often exist as complicated mixtures m plants and are very difficult to purify. The fact that DCCC works well with solvents of high polarity has meant that this technique lends itself particularly well to the separation of tannins. Okuda et al. [15] reported the isolation of new ellagitannins by means of DCCC [n-BuOH-n-PrOH-H,O (2 + 1 + 3) ascending mode], followed by Sephadex LH-20 gel filtration. Anthraquinones (sennosides) have been isolated from rhubarb [16] and anthraqumone glycoside diastereoisomers have been separated by DCCC. The choice of solvent [CHCl,-MeOHH,O (7 + 13 + S)] for the separation of the diastereoisomers aloin A and aloin B of barbalom (13) was governed by consideration of their behaviour on TLC, and LC determination of their partition coefficients [17]. DCCC has been used for the examination of anthraquinones in cell sus-

LIQUID-LIQUID

PARTITION

CHROMATOGRAPHY

IN NATURAL

PRODUCT

ISOLATION

67

pension cultures. In the case of cultures of Morrnda citr:foha (Rubiaceae), both anthraquinone aglycones and glycosides have been separated by DCCC, thus providing an example whereby weakly polar substances are also chromatographed with an aqueous solvent system. Extraction of the cells was carried out with benzene, tetrahydrofuran (THF) and methanol. The benzene extract was subjected to DCCC [n-C,H,,-EtOH-HzOEtOAc (5 + 4 + 1 + 2) ascending mode] and the weakly polar anthraquinones were eluted with the mobile phase. DCCC of the THF extract, under the same conditions, gave the anthraquinone aglycones in the mobile phase. The remaining stationary phase, containing the anthraquinone glycosides, was rechromatographed by DCCC using CHCl,-MeOH-Hz0 (5 + 5 + 3) in the descending mode, in order to separate the glycosides. Further anthraquinone glycosides were obtained from the methanol extract by DCCC with EtOAc-n-PrOH-H,O (7 + 3 + 9) in the ascendmg mode [18]. Ho =I
0 HOCH2 HO

RO
,4

R =Glc-GlcI I Rha Rha R = Xyl-Glc-GlcRka

,e

16

Gk-GkRha

R 1;
H

OH
CHZOH

OH

OH 13

Saponins. These represent a very large group of complex natural products, often with important biological activities. Interest in these glycosidic compounds is growing rapidly, especially as the introduction of modern separation techniques such as DCCC has greatly simplified the problems associated with their isolation. For example, molluscicidal triterpene saponins from Hedera helix (Araliaceae), Lonicera mgra (Caprifoliaceae) and Gundefia tournefortu (Asteraceae) [5] and a closely related mixture of molluscicidal steroid saponins (14-16) from the roots of Balamtes aegyptiaca (Zygophyllaceae) have been isolated. Separation of the steroid saponins by DCCC [CH,Cl,MeOH-H,O (8 + 13 + 7), ascending mode] was much quicker and easier than by open-column chromatography and preparative-scale TLC [19].

Marine organisms have furnished a large number of saponins, among which are the steroid glycoside sulphates. DCCC has enabled the isolation of pure products from very complex mixtures, using solvents such as CHCl,-MeOH-H,O (7 + 13 + 8) and EtOAc-n-BuOH-H,O systems. In one case, a cholest-9(11)-en-23-one glycoside sulphate (17) was isolated from the starfish Luidra macufata via a preliminary purification by DCCC on 10 mm i.d. columns, with n-BuOH-saturated H,O as the stationary phase and H,O-saturated n-BuOH as the mobile phase [ll]. Dlterpenes. Weakly polar diterpenes of the phorbol, ingenol and ingol classes have been successfully separated by DCCC. Liquid-liquid partition techniques are of importance in this work because these substances are often very unstable

NaO,SO

R=(lu~ -Xyl-QUIFL Fllc 17 al,

lQu~=qu~novose)

68

K HOSTETTMANN

AND

MARSTON

J-u
OH
OH 1218 R = 6-H R = o-H

chamomile oil (Matrzcarza chamomzlla, Asteraceae) were obtained with the solvent system &Hi,EtOAc-CH,NO,-MeOH (9 + 2 + 2 + 3) m the ascending mode, followed by n-C,H,,-EtOAcCH,NO,-MeOH (30 + 5 + 15 + 5) in the ascending mode [24].

z E

a6-

19

ji

Volume

[ml]

Fig 3 DCCC of fractionated n-C,H4-Et+n-PrOH-95%EtOH-Hz0 the descendmg mode I: solvent slon from [20] )

011 (510 mg), usmg (4+8+3+5+4) m front. (Repnnted with perrms-

croton

CENTRIFUGAL

PARTITION

CHROMATOGRAPHY

prone to autoxidation. DCCC has also proved capable of resolving very closely related substances, as can be shown by the separation of phorbol (18) the parent alcohol of the potent tumour promoter 12-O-tetradecanoyl phorbol-13acetate, from 4a-phorbol (19) with n-C,H,,Et,O-n-PrOH-95% EtOH-H,O (4 + g + 3 + 5 + 4) as solvent, in the descending mode (Fig. 3). A similar solvent system [ n-C,H,,-Et,O-n-PrOHEtOH-H,O (7 + 16 + 6 + 10 + S), ascending mode] has been used for the separation of ingenol esters from Euphorbza hermentzana (Euphorbiaceae) [21] and E canarzenszs [22]. Non-aqueous systems. Another method of separating apolar or weakly polar compounds by DCCC (especially those which give solubihty problems) is to use non-aqueous solvent systems. For example, a mixture of betulinic acid, betuhn, P-amyrm and cholesterol could be separated into its components by DCCC on columns of 2.7 mm 1.d. with the solvent system MeCN-CH,Cl,-nC,H,, (35 + 15 + 50) in the descending mode [23]. Vulpinic acid (20) was isolated directly from an extract of the lichen Letharza uulpzna with MeOH-Me&O-n-C,H,, (4 + 1 + 5) in the descending mode [23] and terpenoid constituents of
and

Apparatus and prznczple of operatzon CPC is a continuous process of non-equilibrium partition of solute between two immiscible phases contained in rotating coils or cartridges. Although CPC (or centrifugal countercurrent chromatography, as it is also known) rehes, like DCCC, on the principle of liquid-liquid partition for the separation and purification of substances, there are several notable differences between the two techniques, including the followmg: a centrifugal force field is applied, allowing much faster separation times; and as it is not necessary for the solvent system to form droplets, a wider range of two-phase solvent systems is possible. Instruments based on rotating coils can either involve planetary or non-planetary motion about a central axis and, in fact, a whole family of prototypes has been developed, largely due to the work of Mandava and Ito [l]. These include the horizontal flow-through coil-planet centrifuge, the toroidal coil-planet centrifuge, the countercurrent extraction coil-planet centrifuge and the highspeed countercurrent chromatograph. Of these, however, only the technique of high-speed countercurrent chromatography (HSCCC) has been commercialized to any large extent. With all these methods, once the partition coefficients of the solutes are known, it is theoretically possible to predict the location of eluted solute peaks and separations are highly reproducible [25].

LIQUID-LIQUID

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IN NATURAL

PRODUCT

ISOLATION

69

Choice of solvent system The method of TLC screening for solvent systems used in DCCC [4] has been apphed to the search for suitable centrifugal partition chromatographic systems, e.g., in the separation of synthetic peptrdes [26]. For optimum separations with the cartridge CPC method, a solvent system should be chosen in which the R, values of the substances to be separated lie in the range 0.2-0.5 when TLC is carried out with the mobile phase on silica gel plates [27]. For HSCCC, the best R, range is 0.1-0.4. Alternative methods are based on the calculation of partition coefficients by analytical LC [28] or by spectrophotometric measurements [29]. Reversed-phase LC is used to establish the relative concentrations of solutes when they are partitioned between two immiscible phases. The partition coefficient of each component is calculated from the detector response following injection of a solution of the compounds before and after extraction with an immiscible solvent. The cartrzdge system (Sankz CPC) This instrument conststs of a series of cartridges (their number can be varied) located at the circumference of a centrifuge rotor, with their longitudinal axes parallel to the direction of the centrifugal force (Fig. 4) [30]. Each cartridge contains the equivalent of 400 separation channels and connectrons between cartridges are made by narrow-bore Teflon tubes. Whtle the rotor is spin-

Fucose

800 Volume

1200 [ml]

Fig 5. Separation of sugars with the Sanlu mstrument Solvent system, n-BuOH-EtOH-H,O (10 + 2 5 + lo), flow-rate, 4 ml - , rotational speed, 1500 rpm, mobde phase, upper phase. Fzprmted mth pernusslon from [30] )

ning, stationary phase is first pumped into the cartridges, followed by mtroduction of mobile phase at the rotation speed required for the separation. Rotating seals at the upper and lower axes of the centrifuge allow the passage of solvent into the apparatus under pressure. When a steady flow of mobile phase alone leaves the instrument, the sample is introduced and the separation is performed. The rotational speed of the instrument can be varied but although higher speeds generally lead to better resolution, the pumping pressure is increased. Most separations are thus achieved at speeds of ca. 1000 r-pm. The rotor is contained in a thermostated casing so that the temperature of the cartridge installation can be controlled. Another advantage is that either the heavier phase or the lighter phase of a two-phase solvent system can be employed as mobile phase, simply by switching the direction of flow through the instrument. Applzcations. A number of separations with the Sankt CPC system have been reported. Both polar and relatively non-polar substances are compatible wtth the solvent conditions [2,27]. One of the first applications involved the separation of fucose, glucose and sucrose [30]. A sample contammg 100 mg of each sugar was separated with the solvent system n-BuOH-EtOH-H,O (10 + 2.5 + 10) (Ftg. 5). Figure 6 shows a separation of the flavanone hesperetin (21) and the flavonols kaempferol (22) and quercetin (23) by Sankt CPC (Fig. 6b) and

Fig. 4. The Sanka centnfugal chromatograph 1 = Rotary seal Jomt; 2 = connectmg tube, 3 = separation column, 4 = rotor; g = centnfugal force

70

K HOSTETTMANN

AND

MARSTON

~~~~~~~

HO&oH
b
HO

HO
21

22 R=H 23 R=OH

b) al
21 22

23

23 21 10 20 30
Time [h]

Jq
1 2
Tme

[h]

Fig. 6. Countercurrent chromatography of hesperetm (21), kaempferol (22) and quercetm (23). Solvent system, CHCl,-MeOH-Hz0 (33 + 40 + 27); mobrle phase, lower phase; detection, 254 rmr. (a) DCCC separation. Flow-rate, 1.8 ml h-. (b) Sat&t CPC separatron Flow-rate, 2 ml mmW1. Rotational speed, 600 rpm; sample, 6 mg m 1 ml of upper phase and 1 ml of lower phase.

DCCC (Fig. 6a). In both instances the lower phase of the system CHCl,-MeOH-H,O (33 + 40 + 27) was used as the mobile phase and elution was in order of increasing polarity. Whereas DCCC required more than 30 h for complete separation, CPC required only 2.5 h [27].

The Sanki CPC system includes the very important ability to work in the reversed-phase mode. Hence during separation the elution mode can be changed, while simultaneously changing the phases. An example of this very useful adaptation is shown in Fig. 7. With the upper phase of the

a)

b)

Ftg. 7 Sat&r CPC separatron of hesperetm (21), kaempferol (22) and quercetm (23). Condrttons as m Ftg. 6. (a) Mobde phase, upper phase. Flow-rate, 1 ml mu-l (b) Mobile phase, upper phase to 55 mm, then lower phase. Flow-rate, 1 6 ml mm-

LIQUID-LIQUID

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PRODUCT

ISOLATION

71

25

q
CH20H 24 24 R=Ara25 R=Glc26 R=Rha-Ara27 R=Glc-GlcI

26 \/

27

14

20

26

30

II

32

-I

36 Fraction No

40

Fig 8. Sanlu CPC separation of a methanol extract of Hedera hebx (Arahaceae) bernes after n-BuOH-H,O partition Solvent system, CHCl,-MeOH-Hz0 (7 + 13 + 8), mobile phase, lower phase; flow-rate, 1.5 ml mm -l, rotatlonal speed, 700 rpm; sample, 100 mg of extract m 1 ml of lower phase.

CHCl,-MeOH-H,O (33 + 40 + 27) system as mobile phase, a good separation of the three flavonoid aglycones was obtained (Fig. 7a). However, a much more rapid separation was achieved by starting with the upper phase as mobile phase and reversing the phases (i.e., changmg to the lower phase as mobile phase) after elution of quercetin (23) (Fig. 7b). This resulted in reversal of the order of elution of 21 and 22 [27]. The Sanki CPC instrument has also proved valuable for the direct separation of pure compounds from crude plant extracts. In one example, four pure molluscicidal triterpene glycosides were obtained from the methanol extract of Hederu helix (Araliaceae) berries (Fig. 8). The extract was first partitioned between n-butanol and water. The molluscicidal n-butanol residue was then subjected to CPC with the lower layer of the system CHCl,-MeOH-H,O (7 + 13 + 8) as mobile phase. The elution was monitored by TLC and gravimetry. Hederagenin saponins U-27 were

separated within 2 h. Changing the elution mode after fraction 30, such that the upper layer of the solvent system became the mobile phase, enabled two polar saponins to elute in fractions 33 and 35. Fractions 31-34 contained large amounts of polysaccharides [27]. The light petroleum extract of the root bark of Psorospermum febrifugum (Guttiferae) contains a mixture of anthraqumone, anthrone and tetrahydroanthracene pigments, some of which have tumour cell growth-inhibitory activities. Separation of these constituents by multi-step flash chromatography and low-pressure reversed-phase LC resulted m considerable material losses, owing to irreversible adsorption on the sorbents. A single CPC step, however, allowed the direct isolation of three pure compounds (28-30) and a mixture of two anthranoids (31 and a minor component 32), without loss of product (Fig. 9). A lOO-mg sample of crude extract was separated within 4 h using the upper phase of the non-aqueous solvent sys-

72
29 28 I

K HOSTE-ITMANN

AND

MARSTON

OH

OH

moR bl

31+32

31

L
0

R=
I I I

Tlmc

[h]

Fig 9 Sanlu CPC separation of a hght petroleum extract of Psorospermum febnfugum (Guttlferae) n-C,H,,-MeCN-MeOH (40 + 25 + lo), mobde phase, upper phase, flow-rate, 5 5 ml mu-, rotational 254 nm, sample, 100 mg m 1 ml of upper phase and 1 ml of lower phase

root bark Solvent system, speed, 1500 rpm; detection,

tern n-C,H,,-MeCN-MeOH (40 + 25 + 10) as mobile phase [27]. Other applications of the cartridge system include polar products (tannins [31] and flavonoid glycosides [27]) and non-polar products (fatty acids [30], retinals [32], coumarins [27] and naphthoquinones [ 271). The rotating coil system (high-speed countercurrent chromatography) In this technique, the separation column consists basically of a Teflon tube (i.d. 1.6 or 2.6 mm) wrapped as a coil around a spool. The coil is balanced by a counterweight and describes a planetary motion about a central axis (Fig. 10) [33]. In order to prevent leakages, a seal-free system has been developed for mtroduction of solvent mto the rotating coil [34]. Because the coiled column assemblies can be readily interchanged, both analyticaland preparative-scale separations can be carned out. For example, in one commercially available preparative instrument (the multilayer coil separator-extractor, developed by Ito and marketed by P.C., Potomac, MD), the coil consists of a single length of 2.6 mm i.d. PTFE tubing, with a capacity of ca. 350 ml. Rotation speeds for separations are generally of the order of 700-800 rpm.

Concerning the principle of the technique, the motion of the coil causes vigorous agitation of the two solvent phases. A repetitive rmxing and settling process, ideal for solute partitioning, occurs at over 13 times per second [35]. This rapid interchange explains why efficient separations are possible in such a small volume of solvent. However, the mechanism of hydrodynamic distribution of the solvent phases m the coil is not known. A suitable procedure for loadmg the instrument [36] consists first of pumping stationary phase into
inlet

outlet

L
I

ww
n~itii~~~r

I
\

WDa

I I

II

nowing

Ag. 10. Schematic tractor (P C ).

diagram

of the multilayer

cod separator-ex-

LIQUID-LIQUID

PARTITION

CHROMATOGRAPHY

IN NATURAL

PRODUCT

ISOLATION

73

TABLE 2 Selected apphcations of the Ito multtlayer coil separator-extractor Substances separated Plavonotds Plavonol glycosides L~gnan glycosides Tannins Coumarms Coumann glycosides Phenohc acids Alkalotds Solvent system CHCls-MeOH-H,O (4 + 3 + 2) CHCls-MeOH-Ha0 (33 + 40 + 27) EtOAc-H,O + EtOAc-i-BuOH-Hz0 n-GHt,-EtOAc-MeOH-Ha0 (1 + 5 + 4 + 3) n-BuOH-0 1 M NaCl (1 + 1) n-C,H,,-EtOAc-MeOH-Hz0 (18 + 42 + 30 + 30) CHCls-MeOH-Ha0 (13 + 23 + 16) n-C+,H,,-EtOAc-MeOH-H,O (18 + 42 + 30 + 30) n-BuOH-Me,CO-Hz0 (8 + 1+ 10) n-BuOH-0 1 M NaCl Ccl,-MeOH-Ha0 (20 + 20 + 2) CHCls-0 07 M sodium phosphate (1 + 1) Ccl,-MeOH-Ha0 (5 + 4 + 1) Isooctane-EtOAc-MeOH-Ha0 (7 + 3 + 6 + 4) n-QH,,-CHsCN-MeOH (40 + 25 + 10) m the tsolauon of plant-dewed natural products Ref. 38 36 39 40 41 42 43 42 44 45 46 47 48 49 36

Carotenotds Sesqmterpenes Anthranoids

the stationary coil and then introducing mobile phase, after commencing rotation. When the necessary stationary phase has been displaced and mobile phase alone leaves the coil, the sample (ideally in a mixture of the two phases) is introduced via a sample loop. In most instances the instrument can be connected to a UV detector for rapid indication of the progress of the separation. When the lower phase of a biphasic solvent system is the mobile phase, it is introduced via the head end of the coil (i.e., near to the axis of rotation). A recently introduced modification of the pumping procedure now allows variation of phase ratios in the coil and easy reversal of mobile and stationary phases [36]. This is achieved by using separate pumps for the mobile and stationary phases and simultaneous introduction of the two phases into the apparatus. Applzcutrons. The rotating coil technique is finding increasing applications in the purification of natural products. One of the areas of exploitation 1s the preparative separation of antibiotics, exemplified by the isolation of siderochelin A, efrotomycin, pentalenolactone, Bu 2313 B and tirandamycin A and B from crude fermentation extracts of Bacteroldes fragdis and Staphylococcus aurew [37]. Up to 600 mg of sample were introduced for the different separations.

Other applications are especially common m the isolation of plant-derived natural products, as can be seen in Table 2. HSCCC has, for example, proved ideal for the separation of carotenoids. Whereas LC and preparative TLC were unsuitable for the isolation of these pigments from Cochlospermum trnctorium (Cochlospermaceae), cochloxanthin (33) and dihydrocochloxanthin (34) were obtained in one step from a methanol extract of the roots with the coil planet centrifuge [48]. The two-phase solvent system used was Ccl,-MeOHH,O (5 + 4 + 1). The extract (500 mg) was dissolved in 10 ml of 1 + 1 mixture of the two solvent phases before injection. The rotational speed of the instrument was 800 rpm and the flow-rate was 4 ml min-. Resolution of the mixture was achieved within 2 h, with the upper phase as the mobile phase.
CCGH

34
COOH

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K HOSTETTMANN

AND A MARSTON

Purification of the antimalarial agent artemisinin (qinghaosu, 35) from Artemisia annua (Asteraceae) by high-speed countercurrent chromatography has been reported [49]. Aerial parts (123 g) of the plant were extracted with light petroleum and the crude extract was taken up in 15 ml of a 1 + 1 mixture of mobile and stationary phases. The stationary phase was isooctane-ethyl acetate (7 + 3) and the mobile phase methanolwater (6 + 4). Rotation of the coil at 800 rpm and elution of mobile phase at 2 ml min- permitted the separation of artemisinin (35) from artemisitene (36) and arteannuin (37); 123 mg of artermsimn were obtained by this procedure. HSCCC was superior to LC for the isolation of artemisinin because larger amounts could be loaded onto the column and the recovery was better. The alternative method of co-crystallization took much longer.

naceae) by ethanol extraction. The solvent system employed was CHCl,-MeOH-H,O (4 + 3 + 2) with the lower phase as the mobile phase. For the multilayer coil separator-extractor, typically 100 mg of sample were injected, the rotation speed was 800 rpm and the flow-rate was 200 ml h-. Separation was completed within 2.5 h and the partition efficiencies varied from 800 to 530 theoretical plates [38]. Alkaloids are particularly suited to separation by CPC. Quaternary indole alkaloids, for example, are difficult to purify because of their polarity and their interaction with solid support matrices [44,45]. In one application, the alkaloids macusine B (38) and panarine (39) were isolated from a Venezuelan curare [44]. After preliminary purification, they were separated on a multilayer coil separator-extractor fitted with 2.6 mm id. tubing. n-BuOH-Me,CO-H,O (8 + 1 + 10) was used as the solvent system with the lower phase as mobile phase. From a charge of 700 mg, 90 mg of 38 and 100 mg of 39 were obtained.

38 R = CH,OH
39 R = COO-

37

Coumarins [43] and flavonoids [38] have also been separated by HSCCC. With the flavonoids, a comparison was made between the multilayer coil separator-extractor and the horizontal flowthrough coil planet centrifuge. The separator-extractor gave superior results in terms of partition efficiency, separation time and sample loading capacity. The crude flavonoid sample used for CPC separation was prepared from the dried fruits of sea buckthom (Hlppophae rhamnoides; Elaeag-

Finally, an example of a natural products separation will be given which illustrates the versatility of using different pumps for stationary and mobile phases. Not only does this allow gradient operation, but phase reversal is also possible [36]. For the separation of the flavonoids hesperetin (21) kaempferol(22) and quercetin (23) using the solvent system CHCl,-MeOH-H,O (33 + 40 + 27) with the upper phase as mobile phase, the chromatogram illustrated in Fig. lla was obtained. In order to fill the column with exactly 50% of each phase at the beginning of the separation, lower and upper phases were pumped simultaneously at the same flow-rates into the coil. For elution of the sample, mobile phase alone was

LIQUID-LIQUID

PARTITION

CHROMATOGRAPHY

IN NATURAL

PRODUCT

ISOLATION

HOqJ&ocH3 Ho&oH
21 22R=H 23R=OH

bl

23 22 \

/I I 0 1
2

Ad 6 7 8 0 1 2

k 3 I 4

//, 11 5

Time [h] Phase reversal

Time [h]

Fig. 11. Reversed-phase operation of the multdayer co11 separator-extractor. Separation of hesperetm (21), kaempferol (22) and quercetm (23) Solvent system, CHC13-MeOH-H,O (33 + 40 + 27), flow-rate, 3 ml nun-, rotatlonal speed, 700 rpm, detection, 254 nm; sample, 15 mg. (a) Mobile phase, upper phase; (b) mobile phase, upper phase to 70 mm, then lower phase.

pumped. If, after elution of the first peak (quercetin), the phases were reversed and the lower phase was then used as the mobile phase, the chromatogram shown in Fig. llb was obtained. Not only was there an inversion of the elution order of 21 and 22 but also the separation time was considerably decreased [ 361. Conchons Countercurrent chromatography is an important technique for the separation and purification of natural products. As the sample comes into contact only with liquid phases, effects encountered with solid matrices, such as irreversible adsorption, sample loss, denaturation and tailing, are avoided. Tanmns, for example, which present problems of adsorption and hydrolysis during chromatography on solid supports, are very effectively separated by liquid-liquid techmques. Both highly polar and apolar substances can be separated without difficulty. Included in the former

are flavonoid glycosides, anthocyanins, saponins, alkaloids and peptides. Non-aqueous solvent systems have often been used for the separation of apolar products such as triterpenes, steroids, essential oils, anthracene derivatives, naphthoquinones and retinals. In this last category, even isomeric retinals could be resolved on a cartridge instrument. Weakly polar substances (diterpenes, coumarins, etc.) are also efficiently separated with aqueous biphasic systems. The list of applications will grow rapidly because liquidliquid partition is not restricted to any class of natural (or synthetic) product and is equally effective for small ions and macromolecules. Whereas DCCC is a rather slow technique, the recently introduced CPC methods give short separation times of down to 1 h. Sample loads of up to 2 g (and, in the future, perhaps more) are possible and solvent consumption is low (of the order of 1000 ml for a single separation run). In addition to the multilayer coil separator-extractor

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K HOSTEITMANN

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A MARSTON

(P.C.), a high-speed countercurrent chromatograph equipped with two separation columns in series IS currently being developed [50] and further advances in instrumental capabilities will be evident in the near future. The Swiss National gratefully acknowledged Science Foundation for financial support. is

REFERENCES 1 N B Mandava and Y. Ito, Countercurrent Chromatography Theory and Practice, Dekker, New York, 1988. 2 K. Hostettmann, M. Hostettmann and A Marston, Preparative Chromatography Techmques - Apphcations m Natural Product Isolation, Spnnger, Berhn, 1986. 3 K. Hostettmann, M. Hostettmann-Kaldas and K. Nakamsht, J Chromatogr., 170 (1979) 355 4 K Hostettmann, Planta Med , 39 (1980) 1 5 K. Hostettmann, M. Hostettmann and A. Marston, Nat. Prod Rep., 1 (1984) 471 6 J R. Bearder and J MacMillan, m J.R. Lenton (Ed.), Gibberelhns - Chemistry, Physiology and Use, Monograph 5, Bntish Plant Growth Regulator Group, Wantage, 1980, p. 7 B. Domon, M Hostettmann and K Hostettmann, J. Chromatogr , 246 (1982) 133 8 J K Snyder, K. Nakamsm, K Hostettmann and M. Hostettmann, J. Ltq. Chromatogr , 7 (1984) 243 9 T. Tammura, J.J. Pisano, Y. Ito and R.L Bowman, Science, 169 (1970) 54. 10 K. Hostettmann, C. Appoloma, B Domon and M. Hostettmann, J Liq. Chromatogr., 7 (1984) 231. and K Hostettmann, Phytochemtstry, 23 11 D. Schaufelberger (1984) 787. H.C. Krebs, Y. Itakura, R Higucm, K. 12 T Komon, Sakamoto, S. Tagucm and T. Kawasaki, Justus Lteblgs Ann. Chem., (1983) 2092 K Hoktmoto and H. Yamagucm, Chem. 13 A. Numata, Pharm. Bull, 28 (1980) 964. 14 M. Hamburger, M Gupta and K. Hostettmann, Phytochemtstry, 24 (1985) 2689 15 T. Okuda, T Yosmda, M. A&da and K. Yazakt, J. Chem. Sot., Perkm Trans. 1, (1983) 1765 16 Y Ogmara, 0. Inoue, H. Otsuka, K Kawat, T Tammura and S Shlbata, J. Chromatogr., 128 (1976) 218. 17 H -W. Rauwald, Arch. Pharm., 315 (1982) 769. 18 K Inoue, H. Nayesmro, H Inouye and M Zenk, Phytochemtstry, 20 (1981) 1693 Tetrahedron, 38 (1982) 513 19 H.-W Lm and K. Nakamsm, 20 GT Marshall and A.D. Kmghom, J. Chromatogr , 206 (1981) 421

J. Nat. Prod., 21 L J Lm, G T. Marshall and A.D Kmghom, 46 (1983) 723. 22 L.J Lm and A.D. Kmghom, J Agnc Food Chem, 31 (1983) 396. and K Hostettmann, J Chro23 B Domon, M Hostettmann matogr , 246 (1982) 133 24 H Becker, J Reichhng and W.C Hsieh, J Chromatogr, 237 (1982) 307 25 Y. Ito and R.L Bowman, Anal Chem , 43 (1971) 70A J Ltq Chro26 M. Knight, A M Kask and CA. Tammmga, matogr , 7 (1984) 351 27 A Marston, C. Bore1 and K. Hostettmann, J. Chromatogr , 450 (1988) 91. 28 W D Conway and Y. Ito, J Liq. Chromatogr., 7 (1984) 291 29 W.D Conway and Y. Ito, J. Ltq Chromatogr, 7 (1984) 275 30 W Murayama, T Kobayasht, Y. Kosuge, H Yano, Y Nunogakr and K. Nunogakr, J Chromatogr., 239 (1982) 643 31 T Okuda, T Yosmda, T Hatano, K Yazakt, R. Ktra and Y. Ikeda, J Chromatogr , 362 (1986) 375. 32 R.C. Bruemng, F. Dergumt and K. Nakamsm, J Chromatogr , 357 (1986) 340. 33 Y Ito, CRC Cnt. Rev Anal. Chem., 17 (1986) 65 34 Y. Ito and W Conway, Anal. Chem , 56 (1984) 534A 35 Y. Ito, J. Chromatogr., 301 (1984) 377 36 I Slacanm, A Marston and K Hostettmann, J Chromatogr , 482 (1989) 234 37 G.M Bnll, J B. McAlpme and J E. Hochlowskt, J Liq. Chromatogr., 8 (1985) 2259. 38 T.-Y Zhang, X. Hua, R Xiao and S. Kong, J Ltq Chromatogr , 11 (1988) 233. 39 M Vanhaelen and R. Vanhaelen-Fastre, J Liq Chromatogr., 11 (1988) 2969. 40 G R Petttt and D.E. Schaufelberger, J. Nat. Prod., 51 (1988) 1104 41 L.J. Putman and L.G Butler, J. Chromatogr, 318 (1985) 85. 42 K Hostettman, I. Slacamn and A. Marston, unpublished results 43 B.C. van Wagenen, J. Huddleston and J H Cardelhna, J Nat. Prod., 51 (1988) 136. 44 J. Quetm-Leclercq, L. Angenot, L DuPont and N.G Basset, Phytochermstry, 27 (1988) 4002 45 J. Quetm-Leclercq and L. Angenot, Phytochemtstry, 27 (1988) 1923. 46 B. Kanyinda, B. Dtallo, R Vanhaelen-Fastre and M. Vanhaelen, Planta Med , 55 (1989) 394 47 T.-Y. Zhang, D-G C~I and Y. Ito, J. Chromatogr., 435 (1988) 159 48 B Dtallo and M Vanhaelen, J Liq Chromatogr , 11 (1988) 227. 49 N. Acton, D.L KIayman, I J Rollman and J F Novotny, J. Chromatogr., 355 (1986) 448. 50 Y. Ito and F.E. Chou, J. Chromatogr , 454 (1988) 382.