Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor

MODES OF CHROMATOGRAPHY The exact mode of chromatography operating in a given application is determined principally by the nature of the packing, though it must be appreciated that, while there may be one dominant mechanism, the modes are not mutually exclusive. The exact mode of chromatography operating in a given application is determined principally by the nature of the packing, though it must be appreciated that, while there may be one dominant mechanism, the modes are not mutually exclusive.

ADSORPTION CHROMATOGRAPHY The lattice of the common porous adsorbents e.g. alumina and silica, is terminated at the surface with polar hydroxyl groups which provide the means for the surface interactions with solute molecules (Alumina has additional structural features which can influence solute retention; these will be discussed later in the chapter) The lattice of the common porous adsorbents e.g. alumina and silica, is terminated at the surface with polar hydroxyl groups which provide the means for the surface interactions with solute molecules (Alumina has additional structural features which can influence solute retention; these will be discussed later in the chapter)

ADSORPTION CHROMATOGRAPHY The eluant systems used in the adsorption chromatography are based on non polar solvents, commonly hexane, containing small amount of polar additive, such as 2- propanol. When the sample is applied, solute molecules with polar functionality will bond to the active sites on the packing; they will subsequently be displaced by the polar modifier molecules of the eluant, as the chromatogram is developed and will pass down the column to be re – absorbed on fresh sites The eluant systems used in the adsorption chromatography are based on non polar solvents, commonly hexane, containing small amount of polar additive, such as 2- propanol. When the sample is applied, solute molecules with polar functionality will bond to the active sites on the packing; they will subsequently be displaced by the polar modifier molecules of the eluant, as the chromatogram is developed and will pass down the column to be re – absorbed on fresh sites

ADSORPTION CHROMATOGRAPHY The ease of displacement of solute molecules will depend on their relative polarities. More polar molecules will be adsorbed more strongly and hence will elute more slowly from the column. A system may be used. It was the type of apparatus first used for chromatographic separation of the kind familiar today. The ease of displacement of solute molecules will depend on their relative polarities. More polar molecules will be adsorbed more strongly and hence will elute more slowly from the column. A system may be used. It was the type of apparatus first used for chromatographic separation of the kind familiar today.

Solvents As already indicated the solvent plays an active part in the adsorption process and competes with the sample molecules for active sites on the adsorbent. Thus the stronger the binding of solvent molecules the greater the amount of the time the solute molecules spend in the mobile phase, and hence the faster they are eluted. Retention is therefore not so much influenced by sample solubility in the eluent as by the strength of solvent adsorption. It is advisable for a given application to choose an initial solvent of indifferent eluting power so that stronger agents can be tried successively. "Strength" of the eluting agent means the absorbability on the column packing. Generally, for polar adsorbents such as alumina and silica gel, the strength of adsorption increases with the polarity with the adsorbate. For carbon the order is reversed. As already indicated the solvent plays an active part in the adsorption process and competes with the sample molecules for active sites on the adsorbent. Thus the stronger the binding of solvent molecules the greater the amount of the time the solute molecules spend in the mobile phase, and hence the faster they are eluted. Retention is therefore not so much influenced by sample solubility in the eluent as by the strength of solvent adsorption. It is advisable for a given application to choose an initial solvent of indifferent eluting power so that stronger agents can be tried successively. "Strength" of the eluting agent means the absorbability on the column packing. Generally, for polar adsorbents such as alumina and silica gel, the strength of adsorption increases with the polarity with the adsorbate. For carbon the order is reversed.

Solvents One of the first elutropic series was by trapped was recorded by Trapp, who found that the eluting power of series of solvents for substances adsorbed in columns such as silica gel decreased in order One of the first elutropic series was by trapped was recorded by Trapp, who found that the eluting power of series of solvents for substances adsorbed in columns such as silica gel decreased in order Pure water > methanol > ethanol > propanol > acetone > ethyle acetate > diethyle ether >chloroform >dichloromethane >benzene > toluene > trichloroethylene >carbon tetrachloride >cyclohexane >hexane. Pure water > methanol > ethanol > propanol > acetone > ethyle acetate > diethyle ether >chloroform >dichloromethane >benzene > toluene > trichloroethylene >carbon tetrachloride >cyclohexane >hexane. This order is also the order of decreasing dielectric constant. The elueting power of the different solvents has been studied by Williams et al., who found that on an active carbon column the eluting power for amino acids and saccharaides decreased in the order This order is also the order of decreasing dielectric constant. The elueting power of the different solvents has been studied by Williams et al., who found that on an active carbon column the eluting power for amino acids and saccharaides decreased in the order

Solvents ethyle acetate > diethyle ether > > propanol > ethanol > methanol > Pure water ethyle acetate > diethyle ether > > propanol > ethanol > methanol > Pure water This order is of increasing polarity or decreasing chain length of homologues. This order is of increasing polarity or decreasing chain length of homologues. The reverse order was true for alumina and silica gel. The reverse order was true for alumina and silica gel. Practically speaking the solvent is the controlling variable in adsorption chromatography. With the aid of the above series it is possible to select a solvent or solvent mixture with the appropriate eluting power. It should be appreciated that the eluting power of a solvent can be markedly affected by the presence of small amounts of impurity, e.g. methanol in benzene, and hence the purity of the solvents used should be as high as possible. If necessary, further purification can be achived by running the solvent through a column of adsorbent to be used. Practically speaking the solvent is the controlling variable in adsorption chromatography. With the aid of the above series it is possible to select a solvent or solvent mixture with the appropriate eluting power. It should be appreciated that the eluting power of a solvent can be markedly affected by the presence of small amounts of impurity, e.g. methanol in benzene, and hence the purity of the solvents used should be as high as possible. If necessary, further purification can be achived by running the solvent through a column of adsorbent to be used.