1.3.1 Preliminary Purification

Once a suitably polar plant extract is obtained, a preliminary cleanup is advantageous. The classical method of separating phenolics from plant extracts is to precipitate with lead acetate or extract into alkali or carbonate, followed by acidification. The lead acetate procedure is often unsatisfactory since some phenolics do not precipitate; other compounds may co- precipitate and it is not always easy to remove the lead salts.

Alternatively, solvent partition or countercurrent techniques may be applied. In order to obtain an isoflavonoid-rich fraction from Erythrina species (Leguminosae) for further puri­fication work, an organic solvent extract was dissolved in 90% methanol and first partitioned with hexane. The residual methanol part was adjusted with water to 30% and partitioned with t-butyl methyl ether-hexane (9:1). This latter mixture was then chromatographed to obtain pure compounds.18

A short polyamide column, a Sephadex LH-20 column, or an ion exchange resin can be used. Absorption of crude extracts onto Diaion HP-20 or Amberlite XAD-2 (or XAD-7) columns, followed by elution with a methanol-water gradient, is an excellent way of prepar­ing flavonoid-rich fractions.

1.3.2 Preparative Methods

One of the major problems with the preparative separation of flavonoids is their sparing solubility in solvents employed in chromatography. Moreover, the flavonoids become less soluble as their purification proceeds. Poor solubility in the mobile phase used for a chroma- tographic separation can induce precipitation at the head of the column, leading to poor resolution, decrease in solvent flow, or even blockage of the column.

Other complications can also arise. For example, in the separation of anthocyanins and anthocyanin-rich fractions, it is advisable to avoid acetonitrile and formic acid — acetonitrile is difficult to evaporate and there is a risk of ester formation with formic acid.

There is no single isolation strategy for the separation of flavonoids and one or many steps may be necessary for their isolation. The choice of method depends on the polarity of the compounds and the quantity of sample available. Most of the preparative methods available are described in a volume by Hostettmann et al.7

Conventional open-column chromatography is still widely used because of its simplicity and its value as an initial separation step. Preparative work on large quantities of flavonoids from crude plant extracts is also possible. Support materials include polyamide, cellulose, silica gel, Sephadex LH-20, and Sephadex G-10, G-25, and G-50. Sephadex LH-20 is recom­mended for the separation of proanthocyanidins. For Sephadex gels, as well as size exclusion, adsorption and partition mechanisms operate in the presence of organic solvents. Although methanol and ethanol can be used as eluents for proanthocyanidins, acetone is better for displacing the high molecular weight polyphenols. Slow flow rates are also recommended. Open-column chromatography with certain supports (silica gel, polyamide) suffers from a certain degree of irreversible adsorption of the solute on the column.

Modifications of the method (dry-column chromatography, vacuum liquid chromatog- raphy, VLC, for example) are also of practical use for the rapid fractionation of plant extracts. VLC with a polyamide support has been reported for the separation of flavonol glycosides.19

Preparative TLC is a separation method that requires the least financial outlay and the most basic equipment. It is normally employed for milligram quantities of sample, although gram quantities are also handled if the mixture is not too complex. Preparative TLC in conjunction with open-column chromatography remains a straightforward means of purify­ing natural products, although variants of planar chromatography, such as centrifugal TLC,7 have found application in the separation of flavonoids.

Other combinations are, of course, possible, depending on the particular separation problem. Combining gel filtration or liquid-liquid partition with liquid chromatography (LC) is one solution. Inclusion of chromatography on polymeric supports7 can also provide additional means of solving a difficult separation.

Several preparative pressure liquid chromatographic methods are available. These can be classified according to the pressure employed for the separation:

•           High-pressure (or high-performance) LC (>20 bar/300 psi)

•           Medium-pressure LC (5 to 20 bar/75 to 300 psi)

•           Low-pressure LC (<5 bar/75 psi)

•           Flash chromatography (ca. 2 bar/30 psi) High-Performance Liquid Chromatography

HPLC is becoming by far the most popular technique for the separation of flavonoids, both on preparative and analytical scales. Improvements in instrumentation, packing materials, and column technology are being introduced all the time, making the technique more and more attractive.

The difference between the analytical and preparative methodologies is that analytical HPLC does not rely on the recovery of a sample, while preparative HPLC is a purification process and aims at the isolation of a pure substance from a mixture.

Semipreparative HPLC separations (for 1 to 100 mg sample sizes) use columns of internal diameter 8 to 20 mm, often packed with 10 mm (or smaller) particles. Large samples can be separated by preparative (or even process-scale) installations but costs become correspond­ingly higher.

Optimization can be performed on analytical HPLC columns before transposition to a semipreparative scale.

The aim of this chapter is not a detailed description of the technique and instrumentation but to show applications of HPLC in the preparative separation of flavonoids. Some repre­sentative examples are given in Table 1.1. In a 1982 review of isolation techniques for flavonoids,3 preparative HPLC had at that time not been fully exploited. However, the situation is now very different and 80% of all flavonoid isolations contain a HPLC step. Approximately 95% of reported HPLC applications are on octadecylsilyl phases. Both isocratic and gradient conditions are employed. Medium-Pressure Liquid Chromatography

The term "medium-pressure liquid chromatography'' (MPLC) covers a wide range of column diameters, different granulometry packing materials, different pressures, and a number of


Preparative Separations of Flavonoids by HPLC


Phenolics from

Picea abies Chalcones from Myrica serrata

Flavones from

Tanacetum parthenium Flavone glycosides from Lysionotus pauciflorus Flavonoid glucuronides from

Malva sylvestris Flavonol galloyl-glycosides from

Acacia confusa Flavanones from

Greigia sphacelata Prenylated flavonoids from

Anaxagorea luzonensis Prenylated isoflavonoids from

Erythrina vogelii Biflavones from

Cupressocyparis leylandii Anthocyanin glycosides

Proanthocyanidins and flavans from Prunus prostrata


Nucleosil 100-7Ci8

250 x 21 mm LiChrosorb Diol

7 mm, 250 x 16 mm Nucleosil 100-7C18

250 x 21 mm LiChrospher RP-18

250 x 25 mm LiChrosorb RP-18

250 x 10 mm Spherisorb ODS-2

5 mm, 250 x 16 mm Hyperprep ODS 250 x 10 mm LiChrospher Diol

5 mm, 250 x 4.6 mm Asahipack 0DP-90

10 mm, 300 x 28 mm mBondapak C1810 mm,

100 x 25 mm LiChrospher RP-18

7 mm, 250 x 10 mm Spherisorb ODS-2

10 mm, 250 x 10 mm Eurospher 80 RP-18 7 mm, 250 x 16 mm


Me0H-H20, gradient

Me0H-H20, 55:45

Me0H-H20, 76:24

CH3CN-H20, 3:7

CH3CN-H20, 1:4


205:718:62:15 CH3CN-H20, gradient

Hexane-Et0Ac, 7:3

CH3CN-H20, 45:55

Me0H-H20, isocratic

Me0H-H20, 72:28

Me0H-5% HC00H

CH3CN-H20 (+0.1% TFA), 1:4, 3:17













commercially available systems. In its simplest form, MPLC is a closed column (generally glass) connected to a compressed air source or a reciprocating pump. It fulfills the require­ment for a simple alternative method to open-column chromatography or flash chromatog- raphy, with both higher resolution and shorter separation times. MPLC columns have a high loading capacity — up to a 1:25 sample-to-packing-material ratio32 — and are ideal for the separation of flavonoids.

In MPLC, the columns are generally filled by the user. Particle sizes of 25 to 200 mm are usually advocated (15 to 25, 25 to 40, or 43 to 60 mm are the most common ranges) and either slurry packing or dry packing is possible. Resolution is increased for a long column of small internal diameter when compared with a shorter column of larger internal diameter (with the same amount of stationary phase).33 Choice of solvent systems can be efficiently performed by TLC34 or by analytical HPLC. Transposition to MPLC is straightforward and direct.35

Some applications of MPLC to the separation of flavonoids are shown in Table 1.2. Centrifugal Partition Chromatography

Various countercurrent chromatographic techniques have been successfully employed for the separation of flavonoids.7 Countercurrent chromatography is a separation technique that relies on the partition of a sample between two immiscible solvents, the relative propor­tions of solute passing into each of the two phases determined by the partition coefficients of the components of the solute. It is an all-liquid method that is characterized by the absence of a solid support, and thus has the following advantages over other chromatographic techniques:

•           No irreversible adsorption of the sample

•           Quantitative recovery of the introduced sample

•           Greatly reduced risk of sample denaturation


Separation of Flavonoids by Medium-Pressure Liquid Chromatography


Chalcones from Piper aduncum Flavonoids from

Sophora moorcroftiana Flavonol glycosides from Epilobium species

Dihydroflavonoid glycosides from Calluna vulgaris

Prenylflavonoid glycosides from

Epimedium koreanum Prenylated isoflavonoids from Erythrina vogelii

Biflavonoids from Wikstoemia indica


Silica gel

800 x 36 mm RP-18

20 mm RP-18 15-25 mm 460 x 26 mm Polyamide SC-6 460 x 26 mm RP-18 20-40 mm 460 x 15 mm RP-8

460 x 26 mm RP-18 15-25 mm

500 x 40 mm RP-18

300 35 mm



99:0.4:0.3:0.3 MeOH-H2O, 3:1

MeOH-H2O, 35:65

Toluene-MeOH MeOH-H2O

MeOH-H2O, 2:3 MeOH-H2O, 58:42, 60:40

MeOH-H2O, 55:45 ! 95:5









•           Low solvent consumption

•           Favorable economics

It is obvious, therefore, that such a technique is ideal for flavonoids, which often suffer from problems of retention on solid supports such as silica gel and polyamide.

Countercurrent distribution, droplet countercurrent chromatography, and rotation locu- lar countercurrent chromatography are now seldom used but CPC, also known as centrifugal countercurrent chromatography, finds extensive application for the preparative separation of flavonoids. In CPC, the liquid stationary phase is retained by centrifugal force instead of a solid support (in column chromatography). Basically, two alternative designs of apparatus are on the market43: (a) rotating coil instruments; (b) disk or cartridge instruments.

Although most CPC separations are on a preparative scale, analytical instruments do exist.44 However, these are mostly used to find suitable separation conditions for scale-up.

There are numerous examples of preparative separations of flavonoids7,45 and some are listed in Table 1.3.

An example of the separation of flavonoid glycosides by CPC is shown in Figure 1.1. The leaves of the African plant Tephrosia vogelii (Leguminosae) were first extracted with dichlor- omethane and then with methanol. Methanol extract (500 mg) was injected in a mixture of the two phases of the solvent system and elution of the three major glycosides was achieved within 3 h.58

The technique of CPC was also employed as a key step in the purification of 26 phenolic compounds from the needles of Norway spruce (Picea abies, Pinaceae). An aqueous extract of needles (5.45 g) was separated with the solvent system CHCl3-MeOH-i-PrOH-H2O (5:6:1:4), initially with the lower phase as mobile phase and then subsequently switching to the upper phase as mobile phase. Final purification of the constituent flavonol glycosides, stilbenes, and catechins was by gel filtration and semipreparative HPLC.20


Separation of Flavonoids by Centrifugal Partition Chromatography


Solvent System


Flavonoids from Hippophae rhamnoides

CHCl3-MeOH-H2O, 4:3:2


Flavonol glycosides from Vernonia galamensis

CHCl3-MeOH-n-BuOH-H2O, 7:6:3:4


Flavonol glycosides from Picea abies

CHCl3-MeOH-i-PrOH-H2O, 5:6:1:4


Flavonol glycosides from

n-BuOH-EtOH-H2O, 4:1.5:5


Polypodium decumanum

CHCl3-MeOH-n-BuOH-H2O, 10:10:1:6


Flavone C-glycosides from Cecropia lyratiloba

CHCl3-MeOH-H2O, 46:25:29



EtOAc-n-BuOH-MeOH-H2O, 35:10:11:44


Biflavonoids from Garcinia kola

n-Hexane-EtOAc-MeOH-H2O, 2:8:5:5


Isoflavones from Astragalus membranaceus

EtOAc-EtOH-n-BuOH-H2O, 15:5:3:25



EtOAc-EtOH-H2O, 5:1:5


Isoflavones from Glycine max

CHCl3-MeOH-H2O, 4:3:2



CHCl3-MeOH-n-BuOH-H2O, 8:6:1:4


Anthocyanidins from Ricciocarpos natans

n-Hexane-EtOAc-n-BuOH-HOAc-HCl 1%, 2:1:3:1:5


Proanthocyanidins from

EtOAc-n-PrOH-H2O, 35:2:2


Stryphnodendron adstringens



Proanthocyanidins from Cassipourea gummiflua

n-Hexane-EtOAc-MeOH-H2O, 8:16:7:10


Anthocyanins from plants

n-BuOH-TBME-CH3CN-H2O, 2:2:1:5


Polyphenols from tea

n-Hexane-EtOAc-MeOH-H2O, 3:10:3:10



0 1 2 3 * 4 5~

Time (h)

FIGURE 1.1 Separation of flavonol glycosides from Tephrosia vogelii (Leguminosae) with a Quattro CPC instrument. Solvent system, CHCl3-Me0H-Et0H-H20 (5:3:3:4); mobile phase, upper phase; flow-rate, 3 ml/min; detection, 254 nm; sample, 500 mg Me0H extract. (From Sutherland, I.A., Brown, L., Forbes, S., Games, G., Hawes, D., Hostettmann, K., McKerrell, E.H., Marston, A., Wheatley, D., and Wood, P., J. Liq. Chrom. Relat. Technol., 21, 279, 1998. With permission.)

Four pure isoflavones were obtained from a crude soybean extract after CPC with the solvent system CHCl3-Me0H-H20 (4:3:2) (Figure 1.2). The isoflavones were isolated in amounts of 5 to 10 mg after the introduction of 150 mg of sample.52

A combination of gel filtration, CPC, and semipreparative HPLC was reported for the isolation of eight dimeric proanthocyanidins of general structure 1 from the stem bark of Stryphnodendron adstringens (Leguminosae). The CPC step involved separation with the upper layer of Et0Ac-n-Pr0H-H20 (35:2:2) as mobile phase.54





Time (h)



1          R1 = H, R2=OCH3, R3=H (glycitein)

2          R1 = H, R2=H, R3=H (daidzein)

3          R1= Glc6-Ac, R2=H, R3=OH (acetylgenistin)

4          R1= Glc6-Ac, R2=H, R3=H (acetyldaidzin)

FIGURE 1.2 Separation of a crude soybean extract with a multilayer CPC instrument. Solvent system, CHCl3-Me0H-H20 (4:3:2); mobile phase, lower phase; flow rate, 2 ml/min; detection, 275 nm; sample, 150 mg. (From Yang, F., Ma, Y., and Ito, Y., J. Chromatogr., 928, 163, 2001. With permission.)





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