thin- layer chromatography

thin- layer chromatography
introduction
the technique of tlc was first introduction by izmailov and shraiber in 1938 . attempts were made by kirchnen in 1950 who carried out adsorption chromatography on impregnated filter paper an later on glass – fibre paper coated with silicic acid or alumina. stahl was the who introduced TLC as a procedure for analytical adsorption chromatography.
TLC is similsr to paper chromatography , instead of paper, a thin layer of inert material such as al2O3, mgO or sio2 is used as the substrates. other names for TLC are drop, strip, spread layer , surface chromatography an open column chromatography.
superiority of TLC over other chromatography technique
TLC is superior to either papr or column chromatography in the following aspects:
1. it requires simples equipment.
2. development time is short compared to paper and column chromatography which requires several hours or days
3. there is a wide choice of stationary phases. it can be employed for adsorption partition or lon exchange chromatography.
4. early recovery of separated components . it is easy to remove the powdery coating of plates. the spot or zone may be removed quantitatively and required compound dissolved in a suitable solvent is determined by calorimetric or spectrophotometric analysis.
5. effective resolution can be achieved by paper chromatography.
6. under uv light, detection of fluoresuence compounds are easier than on paper chromatography.
7. extremely sharp spot are obtained in TLc. the increase in sensitivity is of the order of 10 to 100 folds as compared to that of paper chromatography.
8. corrosive reagent like sulphuric or chromic acid can be used which is not possible in paper chromatography
experimental techniques
1.Adsorbent –the governing factors for the selection of the adsorbent depends on the solubility, nature and the innertness of the substance to be separated. the following tables contains the details of generally used adsorbents.
Adsorbent acidic or basis activity separatory mechanism compound to be separated
silica gel acidic active adsorption partition acidic and neutral subsyance
alumina basic active adsorption partition basis and neutral
kilesleghur neutral inactive partition strongly hydrophilic substaces
cellulose powder neutral none partition water soluble compounds

A particle size of 1-25 microns is usually used for the separations due to the facts that fine grained particle is one of the resons for the enhanced resolution in TLC.
Adsorbents do not adhere very well to the glass plates. various types of binders are added to overcome this problem. calcium sulphate is commonly used as a binder . other binding materials are starch, hydrated silioconoxie, etc. chemically intert fluorescent indicator such as zinc silicate is added in the adsorbent of the precoated plates which helps in the indentification of non fluorescent but uv absorbing subnstances.

2. preparation of TLC plates
the TLC plates consist of thin layers of adsorbent on a base made of glass , aluminium foill or plastic the adventage of glass plates are 1.they can be easily cleaned and 2. resused. the aqueous slurry of adsorbent is applied to the plate and dried. the thickness of the adsorbent is usally 100-300 micron. the various methods of preparing tin layers are as follows.
1.pouring-Ameasured quantity of the slurry is poured on a given size of plate and the slurry is spread uniformly over the surface.
2. Dipping- in this methods TLC plates are prepared by dipping them at a time, back to back in cholorofrom or chloroform methanol slurries of the asorbent.
3. Spreading- the slurry is placed in an applicator. the apparatus consists of two pairs. in the aligning tray the plates are set in a line and spreader takes up the spreaing mixture and applies it uniformly on a thin methods is used to get thin and uniform layer.
4. Precoated plates- Common adsorbents precoated on glass or aries from 0.1 to 0.2 mm
3. activationof adsorbent
the adsorbent is activated by the removal of the liquid completely which is associated with the thin layer by drying the plate for 30 minutes in air and then in oven at 110? for another 30 minutes. heating silica and alumina plates to 150? for 4 hours result in very active layers.

4.purification of silica gel ‘G'layers
iron present in the silica gel G causes distortion of chromatography. it has been removed by the prelimiminary development of the coated and air dried plates with methanol-conc.HCl.(9:1 v/v). the lron moved with the solvent front to the upper edges of the plate. the plates are again and activated at 110?C.
5.Samples application
Microsyringe is used for the sample application for qualitative work . capillary tubes may also be used.
6. Development chamber
the development chamber used for paper chromatography can also be used for TLC. the common technique employed in TLC is ascending. the plate is placed in the tank at an angle of 45? the chamber is filled with the the solvent at the bottem upto nearly 1mm. three sides of th tank are covered with solvent impregnated paper and the top should be covered tightly with the lid . the chamber should be saturated with solvent vapours to avoid unequal solvent evaporation losses from the developing plate.