Spouted bed extraction

In certain instances, as in the production of annatto powder from the seeds of Bixa orellana, the physical removal of the pigment layer of the seed-coat can yield a less impaired product than that produced by solvent extraction. Such methods can involve the use of a ball mill or a spouted bed unit. A development of the latter, the conical spouted bed extractor, has been investigated for annatto production. Basically it consists of a cylinder tapered at both ends and containing the seeds at the lower end through which a jet of hot air is forced. Seeds and pigment-loaded fine particles are propelled into the space above from whence the seeds fall back to be recirculated and the annatto powder moves to a cyclone from which it is collected. For full details see M. L. Passos et al., Drying Technology, 1998, 16, 1855.

Supercritical fluid extraction

The use of supercritical fluids for the extraction of a range of materials including plant products of medicinal, flavouring and cosmetic interest has, during the last decade, become of increasing economic and research interest.

In 1822, Cagniard de la Tour reported that above a certain temperature, and pressure, single substances do not condense or evaporate but exist as a fluid. Under these conditions the gas and liquid phases both possess the same density and no division exists between the two phases. This is the critical state. For water, the critical conditions for temperature (t_c) and pressure (p_c) are 374°C and 220 atmospheres respectively and for carbon dioxide t_c = 31°C and p_c = 74 atm. In practice conditions somewhat above the critical temperature and pressure for a particular substance are usually used and these supercritical fluids exhibit properties intermediate between those of the liquid and gaseous phases. In phytochemistry these properties can be exploited to maximize the extraction of plant constituents. For industrial purposes supercritical fluid carbon dioxide has an environmental advantage over many common organic solvents and leaves no solvent residues in the product. It also allows a low temperature process and has proved of value for the extraction of labile expensive fragrances and medicinal phytochemicals. To render it more polar a small amount of modifier, e.g. methanol, may be added to the carbon dioxide. The high pressures, and for some substances the high temperatures, involved in supercritical fluid extraction are the principal disadvantages of the technique.

Pioneer work on medicinal plants was carried out by Stahl and coworkers (Planta Med., 1980, 40, 12, and references cited therein). They studied the use of liquefied and supercritical carbon dioxide and liquefied nitrous oxide for the extraction of various plant constituents, including various types of alkaloids, the pyrethrins and the components of chamomile. With pyrethrum flower extract, the content of pyrethrins is substantially higher (up to 50%) than in commercially available petroleum ether extracts. By a two-step precipitation the active ingredients can be raised to up to 60% without decomposition of the thermolabile pyrethrins.

Further examples involving the extraction of phytochemicals with supercritical carbon dioxide follow:

For additional information on the method, consult the ‘Further reading’.

Solid phase microextraction

The method is suitable for some volatile oil-containing drugs. T. J. Betts (Planta Medica, 2000, 66, 193), using methyl polysiloxane solid phase microextraction fibres, has extracted the volatile oil from the headspace above fresh cut eucalyptus leaves (37° for 10 min.). The fibres were then desorbed at 200° for capillary gas chromatography of the oil. Not surprisingly, the oil composition differs from that of steam-distilled oils. The method, coupled with gas chromatography and mass spectrometry, has been recently employed for the analysis of the flowers and essential oils from Lavandula angustifolia cultivated in N.E. Italy (C. Da Porto and D. Decorti, Planta Medica, 2008, 74, 182).

Sublimation

Sublimation may sometimes be possible on the whole drug, as in the isolation of caffeine from tea or for the purification of materials present in a crude extract. Modern equipment employs low pressures with a strict control of temperature.

Distillation

Fractional distillation has been traditionally used for the separation of the components of volatile mixtures; in phytochemistry it has been widely used for the isolation of the components of volatile oils. On a laboratory scale it is not easy by this method to separate minor components of a mixture in a pure state and gas chromatography is now routinely used (q.v.).

Steam distillation is much used to isolate volatile oils and hydrocyanic acid from plant material. The TAS oven (see ‘Thin-layer chromatography’) involves steam distillation on a semi-micro scale for the direct transfer of volatile materials from a powdered drug to a thin-layer plate.

Fractional liberation

Some groups of compounds lend themselves to fractional liberation from a mixture. As an example, a mixture of alkaloid salts in aqueous solution, when treated with aliquots of alkali, will give first the weakest base in the free state followed by base liberation in ascending order of basicity. If the mixture is shaken with an organic solvent after each addition, then a fractionated series of bases will be obtained. A similar scheme can be used for organic acids soluble in water-immiscible solvents; in this case, starting with a mixture of the acid salts, it is possible to fractionally liberate the acids by addition of mineral acids.

Fractional crystallization

A method much used in traditional isolations and still valuable for the resolution of often otherwise intractable mixtures. The method exploits the differences in solubility of the components of a mixture in a particular solvent. Frequently, derivatives of the particular components are employed (picrates of alkaloids, osazones of sugars).

Adsorption chromatography

Of the various methods of separating and isolating plant constituents, the ‘chromatographic procedure’ originated by Tswett is one of the most useful techniques of general application. The use of charcoal for the decolorization and clarification of solutions is well known; coloured impurities are adsorbed by the charcoal and a colourless solution results on filtration. All finely divided solids have the power to adsorb other substances on their surfaces to a greater or lesser extent; similarly, all substances are capable of being adsorbed, some much more readily than others. This phenomenon of selective adsorption is the fundamental principle of adsorption chromatography, the general process of which may be described with reference to one of Tswett’s original experiments.

A light petroleum extract of green leaves is allowed to percolate slowly through a column of powdered calcium carbonate contained in a vertical glass tube. The pigmented contents of the solution are adsorbed on the substance of the column and undergo separation as percolation proceeds. The more strongly adsorbed pigments, xanthophyll and the chlorophylls, accumulate in distinct, characteristically coloured bands near the top of the column, while the less strongly adsorbed pigments, the carotenes, accumulate lower down.

Frequently, complete separation of all the constituents into distinct bands does not result during the first ‘adsorption stage’, but the bands remain crowded together near the top of the column. Such a column may be developed by allowing more of the pure solvent to percolate through the column when the adsorbed materials slowly pass downwards and the separate bands become wider apart. In many cases the process may be rendered more efficient by the use of a different solvent, one from which the substances are less strongly adsorbed. If, for example, light petroleum containing a little alcohol is percolated through the chromatogram obtained in the experiment described above, the bands become wider apart and pass down the column more rapidly than when pure light petroleum is used. As percolation continues, the lower bands reach the bottom of the column and disappear; the pigment is then obtained in the solution leaving the bottom of the column. This process of desorption is termed elution and the solution obtained is the eluate.

It was from such classic experiments of Tswett on the separation of coloured compounds that the term ‘chromatography’ arose and it has remained to describe this method of fractionation although its application to colourless substances is now universal.

Substances are more readily adsorbed from non-polar solvents such as light petroleum and benzene, while polar solvents—alcohol, water and pyridine, for example—are useful eluting media; many substances are adsorbed at one pH and eluted at another.

Various substances may be used as adsorbing materials; alumina is the most common and other materials include kaolin, magnesium oxide, calcium carbonate, charcoal and sugars.

When colourless substances are chromatographed, the zones of adsorbed material are not visible to the eye, although they may, in some cases, be rendered apparent as fluorescent zones when the column is examined under ultraviolet light. Failing this, it becomes necessary to divide the chromatogram into discrete portions and elute or extract each portion separately. Sometimes it is more convenient to collect the eluate from the whole column in fractions for individual examination.

The apparatus required is simple and consists essentially of a vertical glass tube into which the adsorbent has been packed; a small plug of glass wool or a sintered glass disc, at the base of the tube, supports the column. With volatile developing solvents it is usually preferable to use a positive pressure at the head of the column. Numerous modifications of the apparatus are used for large-scale operations, for use with heated solvents and for chromatography in the absence of air or oxygen.

Adsorption chromatography has proved particularly valuable in the isolation and purification of vitamins, hormones, many alkaloids, cardiac glycosides, anthraquinones, etc. It is commonly employed as a ‘clean-up’ technique for the removal of unwanted materials from plant extracts prior to assay.

Thin-layer chromatography with adsorbents such as alumina is an adaptation of the method and is discussed separately in this chapter.

Partition chromatography

Partition chromatography was introduced by Martin and Synge in 1941 for the separation of acetylated amino acids and was first applied to the separation of alkaloids by Evans and Partridge in 1948. The method has now been largely superseded by the more sophisticated HPLC (see below) but it retains the advantage of being inexpensive to set up and operate. The separation of the components of a mixture is, as in counter-current extraction, dependent on differences in the partition coefficients of the components between an aqueous and an immiscible organic liquid.

The aqueous phase is usually the stationary phase and is intimately mixed with a suitable ‘carrier’ such as silica gel, purified kieselguhr or powdered glass and packed in a column as in adsorption chromatography. The mixture to be fractionated is introduced on the column, in a small volume of organic solvent, and the chromatogram is developed with more solvent or successively with different solvents of increasing eluting power. When water is the stationary phase, the solutes undergoing separation travel down the column at different speeds depending on their partition coefficient between the two liquid phases; the use of a buffer solution as aqueous phase widens the scope of the technique, as ionization constants and partition coefficients are exploited in effecting separation.

The separated zones may be located by methods similar to those employed in adsorption chromatography. With water as the aqueous phase, the positions of separated zones of acids or alkalis may be shown by employing a suitable indicator dissolved in the water. This method is clearly not applicable to buffer-, acid- or alkali-loaded columns, and in these cases complete elution (elution development) of the separated zone is often necessary. The eluate is collected in aliquot portions and estimated chemically or physically for dissolved solute. A graph of the analytical figure (titration, optical rotation, optical density, refractive index, etc.) for each fraction of eluate may then be plotted to show the degree of separation of the solutes.

The fractionations obtained in partition chromatography are influenced to a considerable degree by the displacement effect of one solute on another and advantage is taken of this in displacement development, in which the chromatogram is developed with a solution of an acid or a base that is stronger than any in the mixture to be separated. The effect is for the stronger acids or bases to displace the weaker ones, resulting in a rapid clear-cut separation of the constituents. For the elution development of these separated zones it is essential that there is no distortion of the zones, since the front of one band follows immediately on the tail of the preceding less acidic or less basic component.

There have been several theoretical treatments of partition chromatography, all involving certain approximations, since a theory taking into account all known variables would be extremely complicated. For general purposes, one of the most satisfactory treatments of columns loaded with water is that of Martin and Synge, in which the theoretical plate concept of fractional distillation is applied to partition chromatography. In this theory, diffusion from one plate to another is taken as negligible and the partition of solute between two phases is independent of concentration and the presence of other solutes.

Partition chromatography on paper

In 1944 Consden, Gordon and Martin introduced a method of partition chromatography using strips of filter paper as ‘carriers’ for the analysis of amino acid mixtures. The technique was extended to all classes of natural products, and although to a large measure replaced by thin-layer chromatography (TLC), it remains the method of choice for the fractionation of some groups of substances.

The solution of components to be separated is applied as a spot near one end of a prepared filter-paper strip. The paper is then supported in an airtight chamber which has an atmosphere saturated with solvent and water, and a supply of the water-saturated solvent. The most satisfactory solvents are those which are partially miscible with water, such as phenol, n-butanol and amyl alcohol. Either the paper may be dipped in the solvent mixture so that the solvent front travels up the paper (ascending technique) or the trough of solvent may be supported at the top of the chamber, in which case the solvent travels down the paper (descending technique). The BP 2007 gives details of both methods. As the solvent moves, the components also move along the paper at varying rates, depending mainly on the differences in their partition coefficients between the aqueous (hydration shell of cellulose fibres) and organic phases. After the filter-paper strips have been dried, the positions of the separated components can be revealed by the use of suitable developing agents: ninhydrin solution for amino acids; iodine solution (or vapour) or a modified Dragendorff’s reagent for alkaloids; ferric chloride solution for phenols; alkali for anthraquinone derivatives; antimony trichloride in chloroform for steroids and some components of volatile oils; aniline hydrogen phthalate reagent for sugars. The relative positions of the components and the size of the spots depend upon the solvent, and this should be selected to give good separation of the components with well-defined, compact spots. Improved separation of mixtures can often be obtained by adjusting the acidity of the solvent with ammonia, acetic acid or hydrochloric acid or by impregnating the paper with a buffer solution or formamide solution.

For the separation of some substances it is necessary to use a two-dimensional chromatogram: first one solvent is run in one direction, then, after drying of the paper, a second solvent is run in a direction at right angles to the first—this is particularly applicable to mixtures of amino acids.

The ratio between the distance travelled on the paper by a component of the test solution and the distance travelled by the solvent is termed the R_F value and, under standard conditions, this is a constant for the particular compound. However, in practice, variations of R_F often occur and it is desirable to run reference compounds alongside unknown mixtures.

The quantity of substance present determines the size of the spot with any one solvent and can be made the basis of quantitative evaluation. Also, the separated components of the original mixture can be separately eluted from the chromatogram, by treating the cut-out spots with a suitable solvent, and then determined quantitatively by some suitable method—for example, fluorescence analysis, colorimetry or ultraviolet adsorption. Drugs so evaluated include aloes, digitalis, ergot, hemlock, lobelia, nux vomica, opium, rauwolfia, rhubarb, broom, solanaceous herbs and volatile oils.

High-performance liquid chromatography (HPLC)/high-speed LC

HPLC is a liquid column chromatography system which employs relatively narrow columns (about 5 mm diameter for analytical work) operating at ambient temperature or up to about 200°C at pressures up to 200 atm (20 000 kPa).

The columns are costly and it is usual to employ a small precolumn containing a cartridge of packing material to remove adventitious materials which might otherwise damage the main column. Normal flow rates of eluate are 2–5 ml min⁻¹ but can be up to 10 ml min⁻¹, depending on the diameter of the column and the applied pressure. The apparatus is suitable for all types of liquid chromatography columns (adsorption, partition by the use of bonded liquid phases, reversed phase, gel filtration, ion exchange and affinity). The arrangement of such an apparatus, suitable for use with two solvents and giving graded elution, is illustrated in Fig. 17.1. Detection of the often very small quantities of solute in the eluate is possible by continuous monitoring of ultraviolet absorption, mass spectrum, refractive index, fluorescence and electrical conductance; nuclear magnetic resonance can now be added to this list. To improve detection, solutes may be either derivatized before chromatography (this technique can also be used to improve separations) or treated with reagents after separation (post-column derivatization). A transport system for monitoring is commercially available; in this a moving wire passes through the flowing eluate (coating block) and the dissolved solute, deposited on the wire, is pyrolysed and its quantity automatically recorded. It will be noted that, for any particular fractionation, some detector systems would be selective for certain groups of compounds and others would be universal.

Fig. 17.1 Schematic representation of apparatus for high- performanance liquid chromatography utilizing two solvents.