Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor

Physical Methods ► UV detection ► The most common method of location uses an adsorbent layer containing a fluorescent indicator. Commercially available plates uses an indicator which absorbs light at 254nmand re- emits or fluoresces light at the green end of the spectrum; thus the plate when irradiated at 254nm takes on striking green color.

Physical Methods ► If a spot of compound is present which itself absorbs at 254nm, this will quench the fluorescence and the component will show up as dark spot against the green background. While this is not a specific identification the technique has the advantage, since the indicator is insoluble in common solvents and the location is non- destructive, of allowing isolation of the component for subsequent spectroanalytical analysis. This is achieved by scraping the sorbent where the spot is positioned from the plate and extracting with suitable solvent. Removal of solvent under reduced pressure leaves the pure component. Other reagents used fluorescence at 370 nm.

Physical Methods ► Preparative TLC ► Thin – layer chromatography can be scaled up and used for the isolation of large (10-10 ² mg) quantities of pure component. The practice of the technique is similar to that for analytical, qualitative scale work. The main difference lies in the plates used. Almost all preparative scale work is carried out, in the adsorption mode, principally on silica gel plates of varying thickness,1-5mm, and of 20X20cm dimensions.

Physical Methods ► The sample is applied as a streak, either by pasture pipette, syringe or motorized "streak applicator" advantage can be taken of multiple development techniques, which allow efficient separation of markedly different polarities, bands incompletely resolved can be applied to a fresh plate and rechromatographed with a suitable solvent and development procedure. Once development is completed the bands of component can be scrapped off with razor blade or spatula and the components washed off the absorbent with suitable solvent. Plates for preparative chromatography are available with added fluorescent indicator which facilitates non – destructive location of the components. The fluorescent indicator is irreversibly bound to the silica.