Presentation on theme: "Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor"— Presentation transcript:
Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor
PARTITION COLUMN CHROMATOGRAPHY ► In partition chromatography the solid adsorbent is replaced by packing comprising a support material coated with stationary phase. The stationary phase should be insoluble or at the most sparingly miscible in the mobile phase. ► Partition chromatography is a technique which utilizes the ability of solute to distribute itself between the two phases, to an extent determined by its partition coefficient. The basis of this method is that because of differences in the partition coefficients of the various components, the mixture will be resolved.
PARTITION COLUMN CHROMATOGRAPHY ► The stationary phase is supported on a solid which is inert to the substances to be separated. The coated solid is packed in columns as in adsorption chromatography. There is, in fact very little visible difference between the two types of column. The support material must adsorb and retain the stationary phase, it must be exposes as large a surface of it as possible to the flowing phase. It must be mechanically stable and easy to pack into the column when loaded with the stationary liquid, and must not impede the solvent flow.
PARTITION COLUMN CHROMATOGRAPHY ► Needless to say; there is no support which has all these properties to the desired extent. The greatest difficulty (more or less unavoidable when a solid has to be used) is the incursion of adsorption effects. Even if the surface of the support is completely covered with liquid, adsorption effects can still make themselves felt. As complete coverage of the surface is not easy to achieve adsorption may be a major influence on the separation. As far as liquid – phase chromatography on column is concerned it is probably true to say that the division into adsorption and partition methods is of practical, rather than theoretical, significance. The importance of adsorption varies from system to system and is mentioned briefly in connection with the different supports described below. The moving phase in partition chromatography may be a liquid or a gas, and the general principles are the same in each case.
Solvents Solvents ► It is normal to choose a system so that there is a considerable difference between the solvent strength parameters of the mobile and stationary phase, e.g. with water as stationary phase, pentane would be the optimum choice as eluent. However, over a long period of time the stationary phase will be stripped /washed from the column. This is referred to as solvent stripping, which led to the development of chemically – bonded stationary phase for HPLC. This problem can be overcome by pre – saturating the eluent with the stationary phase before it contacts the packing. This can be achieved either by stirring the two phases together until equilibration is achieved or by placing a pre – column at the chromatographic – column inlet.
ION EXCHANGE CHROMATOGRAPHY ► Ion exchange is a process wherein a solution of an electrolyte is brought into contact with an ion exchange resin and active ions on the resin are replaced by the ions (ionic species) of similar charge from analyte solution. ► Action exchanger is one in which the active ions on the ion exchange material and the exchange process involves cations. The polar groups in cations – exchangers are acidic, commonly – SO3H-CO2 H-OH or PO4 H3
ION EXCHANGE CHROMATOGRAPHY ► They are attached to the polymer molecule in a regular way and are accessible to the solution containing the ions to be removed and separated. The polar groups in anion exchangers are tertiary or quaternary ammonium groups (- CH2- NR2- or (- CH2- NR3) and they function in an analogous manner. Anion exchangers are usually supplied in the chloride form rather than hydroxide because of the greater stability of the former. ► Ion- Exchange chromatography on columns was initially restricted to the use of resins, mainly because of their desirable properties such as chemical stability.