Chromatography Dr.Tawfeq A. Al-Howiriny Associate Professor

PLANE CHROMATOGRAPHY ► THIN - LAYER CHROMATOGRAPHY (TLC) ► The idea and fundamentals of using a chromatographic adsorbent in the form of this layer fixed on an inert rigid support seem to have been suggested by Izmailov and Shraiber in ► The apparatus required is relatively inexpensive and the technique is easy to learn and is fast and versatile.

PLANE CHROMATOGRAPHY ► Outline of the method: ► In thin layer chromatography a solution of the sample in a volatile solvent is applied via pipette to the bottom of a uniformly spread over a suitable supporting plate of material such as glass or plastic and dried under standard conditions. When the spot has dried the plate is placed vertically in a suitable tank with its lower edge immersed in the selected mobile phase. The solvent rises by capillary action and an ascending chromatographic separations thus obtained, resolving the sample mixture into discrete spots.

PLANE CHROMATOGRAPHY ► At the end of the run the solvent is allowed to evaporate from the plate and the separated spot are located and identified either by physical methods such as a sight, fluorescence or radiation monitoring, or by treating chemically with a developing reagent ; the method chosen id dictated by the composition of starting mixture.

PLANE CHROMATOGRAPHY ► In its simplest for a TLC plate can be prepared in the laboratory, placed in any suitable size container. And the resultant chromatograph scanned visually. At its most sophisticated there is a large variety of plates, sample application aids, developing chambers, visualization aids and adsorbents commercially available. TLC can be an excellent qualitative and quantitative method.

Comparison of thin – layer with other forms of chromatography ► TLC has a number of advantages: ► Simultaneous analysis of multiple standards and samples can be carried out under identical conditions in a time comparable to HPLC ► Strongly retained compounds in comparison to HPLC forms the most compact chromatographic zones and therefore can be detected with the highest sensitivity. In addition the bonds can be removed and purified ► All components can be located, unlike in HPLC, where highly polar material may be over looked as the peaks are very broad and difficult to discern

Comparison of thin – layer with other forms of chromatography ► In thin layer chromatography, only small amounts of adsorbents and minute samples are needed. The separated spots are located on the plates using a variety visualization technique in the common with paper chromatography, so that normally no collection of fractions is necessary. There is however, no difficulty about the preparative separations, which are achieved by increasing the thickness of the layer and using a higher loading sample. After separation, it is easy to recover an individual substance by scrapping and collecting the part of the layer in which it is adsorbed. The substance can be extracted with suitable solvent.

Comparison of thin – layer with other forms of chromatography ► Compared with paper chromatography, the main advantages of the thin- layer method are greater speed, and in most cases, better resolution. the average time for a 10 cm run in thin- layer chromatography, on silica gel is minutes ( depending on the nature of the mobile phase), whereas the same separations on fast papers might take two hours. Rough qualitative separation on small plates may take as little as five minutes. The better solution arises from the fact that the adsorption in thin- layer chromatography has a higher capacity than the paper chromatography and the particle size of the adsorbent layer material is very small compared to the large cellulose fibers from which the paper matrix is formed. The separated spots therefore retain fairly closely the shape and size of the original applied spot, without the spreading associated with partition chromatography on paper.

Comparison of thin – layer with other forms of chromatography ► This advantage is largely lost when a partition system is used on this layer. A further, and very important, advantage of the adsorption system is that it can be used to separate hydrophobic substances, such as lipids and hydrocarbons, which are difficult to deal with on a paper, even with a reversed phase system. Thin- layer separations have been applied, however, in most fields of organic, and some inorganic chemistry. Location of separated substances on thin layers is done in the same way as it is on the paper, but more reactive reagents for example concentrated sulphuric acid on thin layers, provided that the thin- layer material is an inert substance such as silica gel or alumina.