Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor

Solvents Solvents ► The choice of eluant as in column chromatography is determined by the sorption process employed and by the nature of the sample. The polarity of the solvent typically expressed in an eluetropic series in which they are arranged in order of increasing polarity as indicated by their dielectric constant. ► Generally, a solvent or a solvent mixture of the lowest polarity consistent with a good separation should be employed. Suitable mixing gives mobile phases of intermediate eluting power, but it is best to avoid mixtures of more than two components.

Solvents ► As much as possible, chiefly because more complex mixtures readily undergo phase changes with changes in temperature. When mixtures are used, greater care is necessary over equilibration. The purity of the solvent is of much greater importance in thin- layer than in most other forms of chromatography, because of small amounts of material involved.

Solvents ► Location of separated Substances ► The success of chromatographic separation depends ultimately on the location process. Colored substances are, of course, visible as separate batchesat the end of the run. ► Colorless substances require chemical or physical detection

Solvents ► Chemical Method ► Chemical method of detection involve the application of a derivatising agent, commonly referred to as allocating reagent, or chromatographic reagent, to the TLC plate. The reagent in a suitable solvent is applied as spray to the plate, when a colored derivative is formed in situ. The reagent may be classified as non specific if they produce colored spots with a wide range of compound classes for instance, iodine, sulphuric acid, Rhodanine B and fluorescein -or they may be specific and only react with compounds containing a particular functional groups e.g. dinitrophenylhydrazine for carbonyl compounds. Thus specific chromogenic reagents can be applied successively and judicious choice will not only make the spots visible but will also aid in the component identification

Solvents ► It is frequently necessary to heat the plate after spraying to accelerate the chemical reaction between reagent and components this requires specialized heating chambers to provide uniform conditions for even spot development. An alternative to spraying is to dip the TLC plates into a solution of the reagent to the plate, sample can be lost from the plate and spreading of the spots may occur which leads to a loss in resolution and sensitivity.

Solvents ► As a rule these method of location are times more sensitive on LTC than on paper chromatography with the added advantage that more corrosive agents can be employed. Comprehensive lists of spray reagents for TLC and PC are available from manufacturers and in the chemical literature. ►