Presentation on theme: "Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor"— Presentation transcript:
Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor firstname.lastname@example.org
Documentation Documentation ► It is obvious from the above discussion that any description of chromatographic procedure the fullest possible details should be presented. Not only are R f values important. But the shape and color of spot give valuable information also. ► The most successful method is to photograph the plate and commercial apparatus is available.
Documentation ► 3.1.8 Development ► Once chromatographic plate has been prepared and the samples have been applied to it, it is placed in a suitable chamber with the lower edge immersed in the eluant to a depth of 0.5-1.0 com ► This sandwich cab the be placed in a normal development chamber or into a special trough design to prevent solvent loss
Documentation ► Ascending ► In the ascending technique the TLC plate is positioned in the development tank after it has come to equighlibrium with the solvent; the application of the sample spot should be above the solvent level. The solvent percolates through sorbent material by capillary action moving the components to differing extents, determined by their distribution coefficients Dx, in the direction of the flow of the eluant.
Documentation ► Descending ► The descending technique through more common paper chromatography can be used in TLC. The top of the plate, where the spots are located, has solvent from a trough fed into it via a wick; some solvent of the same composition is placed in the bottom of the tank but the plate is supported above the solvent level. In order to improve the resolution for particularly difficult separations a number of modifications to the above technique have been developed.
Documentation ► Continuous development ► It is not necessary in either ascending or descending techniques to remove the plate once the solvent has traversed the sorbent layer. In ascending chromatography the eluant is allowed to wash off the top of the plate and a continuous flow of solvent is obtained. Descending chromatography readily lends itself to this modification, the eluant simply being allowed to wash off the plate into the bottom of the tank. Descending development has the advantage of quicker solvent flow, due to the action of the capillary and gravity forces on the eluant, though it has the disadvantage of requiring additional equipment and additional expertise to set up. Continuous development can be used to good effect for the resolution of compounds using low polarity solvents.
Documentation ► Stepwise development ► There is no reason why the development process cannot be interrupted, the plate removed from the tank, solvent allowed to evaporate off and the whole sequence repeated. The subsequent elution procedure can be modified; for instance, different solvent systems can be used and the solvents allowed to migrate each to a different extent. The sequential use of series of eluants of differing eluetropic strength can be used for the separation of the mixtures of wide ranging polarity. Depending upon the nature of mixture either an increasing or decreasing series of development may be used.
Documentation ► Multiple development ► The method is particular variant of stepwise development where the same solvent system is used successively.
Documentation ► Two Dimensional development ► Sometimes, particularly in the case of large groups of compounds of similar chemical constitution, such as amino acids, the Rf values are too close together to give good separation using one – dimensional linear development techniques. In these instances, improved resolution can be obtained with two- dimensional development a technique developed by Martin, which employees a second eluent system run at the right angels to the first. The sample is spotted in the normal manner and developed with the edge (AB) of the plate in contact with the first solvent system. the plate is then removed and allowed to dry. It is then developed with a second solvent at right angle step the first, edge (AC) of the plate in the solvent.
Documentation ► Radial Development ► The technique of radial development, sometimes refer to as horizontal chromatography and also known as Rutter Method, involves a slightly different principle from those so far described. In this method the sample spot is applied to the centre of the plate (a disc) and the solvent is supplied through a hole in the plate via a wick which dips into a solvent reservoir. In a variation proposed by Litt & John the plate is placed with the sorbent layer facing downward with its centre in contact with a porous "spring loaded" wick standing in the solvent. As development proceeds the components move out radially forming circles of increasing diameters. There is an inherent advantage here, since there is a concentration effect, as the annular zone are formed, due to the solvent moving the trailing edge faster than the leading edge. Because of this concentration effect the resolution of the components of low Rf with linear develop is much improved.
Documentation ► Gradient Elution ► The technique of gradient elution in which the composition of the mobile phase is modified as the chromatographic development is in progress, has been applied to TLC. The plate stand as usual in one solvent and the second is added slowly, with magnetic stirring. A constant device is necessary.