Presentation on theme: "Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor"— Presentation transcript:
Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor
Sample application ► The sample should be applied to the top of the column as evenly as possible, and in as concentrated as solution of the eluting solvent as possible, avoiding disturbance of the column packing. The top of the packing can be protected with a thin layer of sand, glass wool, or filter paper. When all samples have been adsorbed the void can be filled with solvent and the chromatogram developed. The supply of solvent can be replenished as required.
Sample application ► Elution procedure ► There are three principle elution procedures; isocratic ( from the Greek, isochors meaning equal strength); stepwise or (fractional ); and gradient.the isocratic procedure is the operating of the chromatographic column by allowing a solvent mixture of unvarying composition to run through the column until the separation is complete.
Sample application ► If the separated constituents of the mixture can be observe on the column (either by their color, their reaction with an indicator previously or subsequently applied to the column, or, perhaps, by their fluorescence in ultraviolet light), the development can be stopped. The content of the column can now be extruded and the separated constituents extracted by means of suitable solvents.
Sample application ► Isolation of the bands is facilitated by the use of transparent nylon tubes as the column container. When the separated constituents have been located the whole tube is cut into sections and the separated parts of the column are removed. An alternative and more commonly used method is to allow the column to run until the separated components can be detected in the column effluent (eluate).
Sample application ► The importance of mobile phase flow rate has previously been discussed. If the solvent flow has to be accelerated it is better to apply pressure to the top of the column by means of compressed gas rather than no reduce pressure at the bottom. Reduction of pressure is liable to cause channeling ► By allowing dissolved gas to come out of solution.
Sample application ► Stepwise or fractional elution ► If only one solvent is used ready to elution of only some of the components of the original mixture from the column may result. To remove those which are more firmly held stronger eluting agent will be required. Sometimes it may be necessary to use several different solvents of gradually increasing strength for the successive desorption of different components. This is unknown as stepwise elution.
Sample application ► It has the advantage that sharper separations may be obtained than if only one strongly eluting solvent, capable of moving even the most firmly bound of the components of the mixture, is used- a part from the possibility displacement development. One danger of this technique, however, is that given compound may give rise to more than one peak by appearing in the eluates of successive steps.
Sample application ► Gradient elution ► The technique of gradient elution analysis was first described in detail by Alam, Williams and Tiselius. It involves the uses of continuously changing eluting medium. The effect of this gradient is to elute successively the more strongly adsorbed substance and at the same time to reduce tailing. This means that the chromatographic bands will tend to be more concentrated and thus occupy less of the column.