Counter current Extraction
Advantages:-Saponins do not produce trouble some foams and unwanted oxidation changes are avoided, little preliminary purification of the plant extract is required. And the apparatus is simple to operate and clean.
The disadvantages are the relatively long separation times which is upto several days during which the unstable compounds may decompose and a solution which does not compare with HPLC. The method has been used for the satisfactory sepatation of many group of compounds including Saponins (Triterpenoid, spirostanol, cardioactive ginsenoside bupleureum (saiko Saponins) iridoid and secoiridoid glycosides polyphenols including anthraquinone glycosides alkaloids and sugars.
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 separation of components of a mixture is as in counter current extraction is dependent on differences in the partition co efficient of the components between an aqueous and an immiscible organic liquid. The method has now been largely superseded by the more sophisticated HPLC but it retains the advantage of being in extensive to set up and operate. The aqueous phase is usually the stationary phase and is intimately mixed wit a suitable carrier such s silica gel. Purified Kiesselguhr or powdered glass and packed in a column as in adsorption chromatography. The mixture to be fractionated is introduced on the column in small volume of organic solvent and the chromatogram is developed with more solvent or successively with different solvents of increasing dluting power. When water is in stationary phase the solutes undergoing separation travel down the column at different speeds depending on their partition co-efficient between the 2 liquids phases. The use of a buffer solution as aqueous phase widens the scope of technique as ionization constants and partition coefficient are exploited in effecting the separation.
The separated zones may be located by methods similar to those employed in adsorption chromatography with water as the aqueous phase. The position of separated zones of acids or alkalis may be shown by employing a suitable indicator dissolved in water. This method is not clearly applicable to buffer, acid, bases loaded columns and in these cases complete elution of the separated zones is often necessary. The elute is collected in aquoes portions and estimated chemically or physically for dissolved solute.
The fractions obtained in partition chromatography are influenced to a considerable degree by the displacement effect of one solute another advantage is taken of this in displacement development in which the chromatogram is developed with a solution of acid or base that is stronger than any mixture to be separated. The effect is for the stronger acids or bases to displace the weaker ones, resulting in a rapid cut separation of the constituents for the elution development of these separated zones. It is essential there is no distortion of the zones. Since the front of one band follows immediately on the tail of preceding less acidic or basic component.
Partition chromatography on paper:-
The solution of components to be separated is applied as a spot near one end of an prepared filter paper strip. The paper is then supported in an air tight 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 phemols, n-butanol and amylalcohol. Either 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). As the solvent moves, the components also move along the paper at varying rates, depending upon the difference in their partition co efficient between the aqueous (hydration shell of cellulose fibers) 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 Dragendroff reagents for alkaloids, ferric chloride solution for phenols, alkali for anthraquinone derivatives, antimony tri chloride in chloroform for strriods 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 solbent with ammonia, acetic acid or HCl or by impregnating the paper with a buffer solution or a 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 traveled on the paper by a component of the test solution and the distance traveled by the solvent is termed the RF value and under standard conditions this is a constant for the particular compound. However in practice variations of RF often occur and it is desirable to run reference counpounds along side 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 original mixture can be separately eluted from the chromatogram by treating the cut out spots with a suitable solvent and then determined by some suitable method. For e.g. Fluroscence analysis colorimetry or UV adsorption. Drug so evaluated includes aloes, digitalis, ergot, hamlock, lobellea, Nux-vomice, opium, Rauwalfia, Rhubarb, Solanaceous herbs and volatile oils.
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i am M.Pharma in cognosy and M.Sc in chemistry.