pharmacognosy Separation and Isolation of Constituents.pdf

pharmacognosy Separation and Isolation of Constituents Separation and Isolation of Constituents Content: Introduction 3 Sublimation 4 Distillation 5 Fractional liberation 7 Fractional crystallization 9 Adsorption chromatography 11 Separation and Isolation of Constituents Separation and Isolation of...

pharmacognosy Separation and Isolation of Constituents Separation and Isolation of Constituents Content: Introduction 3 Sublimation 4 Distillation 5 Fractional liberation 7 Fractional crystallization 9 Adsorption chromatography 11 Separation and Isolation of Constituents Separation and Isolation of Constituents The most difficult operation in phytochemical research is the isolation and purification of plant constituents. Although the chemical properties of functional groups and moieties contained in compounds such as acids, aldehydes, phenols and alkaloids can be exploited for their separation from other materials, Such methods might not fractionate components of the same class; it is in this latter area that new techniques are constantly being developed. Separation and Isolation of Constituents 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. Separation and Isolation of Constituents 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. Separation and Isolation of Constituents 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. Separation and Isolation of Constituents Fractional liberation : Some groups of compounds lend themselves to fractional liberation from a mixture. An example, a mixture of alkaloid salts in aqueous solution, when treated with 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. Separation and Isolation of Constituents A similar scheme can be used for organic acids soluble in waterimmiscible 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. Separation and Isolation of Constituents Fractional crystallization : A method much used in traditional isolations and still valuable for the resolution of 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 Separation and Isolation of Constituents 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. Separation and Isolation of Constituents 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 thanothers. 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. Separation and Isolation of Constituents 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. Separation and Isolation of Constituents 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. Separation and Isolation of Constituents 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. Separation and Isolation of Constituents 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. Separation and Isolation of Constituents 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. 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.