Thin layer Chromatography (TLC)-Principle and procedure

Principle of thin layer chromatography

• It is similar to paper chromatography except that a thin (0.25mm) layer of some inert material such as Al₂O₃, MgO, or SiO₂ is used as the substrate instead of the paper.
• A layer of one of these oxides is made from slurry of powder in a suitable inert solvent.
• The slurry is spread evenly over a flat surface (glass) and dried it may be spread manually or mechanically.
• The advantage of using inert substances instead of paper is that more reactive developing reagents, such as strong acids, can be used to detect the compounds without destroying the substrate.
• It is especially useful for the separation and analysis of high molecular weight biochemical compounds.
• A wide variety of mixtures such as amino acids, dyes, food colorings, drugs, sugars, natural products and insecticides may be separated and identified.
• Stationary phase:
  • The most commonly used stationary phases are adsorbents e.g. fine powders of silica gel, alumina, Kieselguhr and cellulose, they are specially prepared for TLC.
  • Many of these are available with a fluorescent compound e.g. ZnS incorporated inorder to facilitate the detection of the resolved components of the mixture when viewed under UV light.
  • Silica gel is slightly acidic whereas alumina is slightly basic but neutral alumina is also available.
  • Silica gel and alumina particles contain hydroxyl groups on their surface which will form hydrogen bond with polar molecules.
  • Alumina is preferred for the separation of weakly polar compounds, but silica gel is preferred for polar compounds such as amino acid and sugars.
  • Other absorbents suitable for special purpose are polyamides, magnesium silicate, calcium silicate, activated charcoal, modified cellulose with ion exchange properties and the various forms of organic gel e.g. Sephadex, Bio-Gel P.
  • Support used for the stationary phase in TLC is a glass plate , aluminium metal plate or plastic strip.
  • Glass plates can be used in a number of sizes ranging from microscope slides to larger plates (20cm X 20cm X 20cm).
  • Binder is essential for good adherence of the adsorbent to the plate.
  • Commonly used binder is gypsum (calcium sulphate) which is incorporated at a level of 10-15%.
  • Starch and certain organic polymers are also used as binders.
  • Pre-coated plates are advantageous over the prepared plates that have uniform and non-fragile coating.
• Mobile phase:
  • The solvent used are listed below in order of increasing polarity, it is known as eluotropic series.
  • Petroleum ether < n-hexane < carbon tetrachloride < toluene < benzene < chloroform < dichloromethane < diethyl ether < n-butanol < isopropanol < acetone < methanol < water
  • Movement of the solute increases with increasing solvent polarity.
  • The solvent is employed in the form of a pool at the bottom of the developing chamber.
  • Usually the solvent or eluant is made to ascend on the plate, hence the name ascending TLC.
  • The solvent travels through the system by capillary action, thus solvent velocity is determined by the nature and packing structure of the adsorbent.
  • The saturation of the development chamber with solvent vapor has a significant effect on solute migration.
  • Specifically, with under-saturation solvent and solute migrations are slow and not smoothly ascending.
  • This problem can be overcome by lining the developing chamber with a filter paper which is then saturated with developing solvent to provide uniform chamber saturation.
  • Selection of the developing solvent is done by preliminary trial runs using microscopic slides or small strips.

Procedure of Thin layer chromatography

Step I: Plate preparation:

• Cleaning – Glass plates must be carefully cleaned with detergent to remove adhering particles rinsed thoroughly with distilled water, placed in a metal rack and dried in an oven.
• The plate should be handled by the edges or by the under-surface which is not to be coated with the adsorbent.
• Failure to take the precaution and grease contamination on the glass surface may result in the formation of poor quality mechanically unstable layer, which is liable to be flaking.

Step II: Adsorbent selection:

• Commonly used adsorbents are silica gel, alumina, cellulose and polyamide.

Step III: Slurry preparation:

• Slurry is prepared by the slow addition with stirring of adsorbent, e.g. silica gel or alumina, to a suitable solvent like water, dichloromethane in a wide mothed capped bottle.
• Too thick or too thin slurries should be avoided.

Step IV: Coating of the TLC plate:

• The supporting plate (glass, metal etc.) should fulfill the following requirements:
  • Uniform thickness
  • Inert to solvent, solute, stationary phase, identification reagents, procedures
  • Sufficient strength to allow vertical development.
• Thin layer may be prepared by pouring, dipping, spraying or spreading the adsorbent slurry over the plate.
• Generally, the best procedure for the preparation of uniform layers or films is the use of the commercial spreader.
• However, the best source of uniform plates may be pre-coated plates that are available from manufacturers and suppliers.

Step V: Activation of TLC plate:

• After the slurry has been spread out evenly the plates are placed horizontally to set for approximately 10 minutes in a fume cupboard.
• The surplus adsorbent is removed from the glass edged by means of razor blade or glass rod.
• The plate is then activated by heating at 110^oC for 1 hour in an oven.
• The drying conditions may vary with the nature of the adsorbent, binder and the solvent.
• After they are dry, the plates should be cooled to room temperature and stored in desiccators until used.
• Cellulose and polyamide plates are allowed to dry at room temperature and are not normally heated, then stored in a dust free cabinet.
• After drying, normal layer thickness is in the range of 150m for analytical and 2mm for preparative TLC system.

Step VI: Sample preparation:

• The mixture (e.g. a mixture of amino acids) to be analysed is dissolved in a suitable solvent (0.5-3%).
• The selected solvent should be volatile for rapid evaporation of the solvent is desirable as this leads to the formation of a small-diameter spot which results in a better separation during chromatographic development process.
• Reference compounds are similarly prepared and applied to the adsorbent on the same plate alongside the mixture spot, this helps more ready interpretation of the chromatogram.

Step VII: Sample application on the TLC plate:

• Wipe any excess adsorbent from the back and edges of the plate.
• Sample should normally be applied about 5mm (for small plates) to 10mm (larger plates) from the edge of the plate.
• However, care should be taken not to immerse the spot in the solvent pool in the development chamber.
• The spots should be separated from each other by at least 10mm for larger plates.
• Sample application is performed by spotting or streaking the thin layer.
• Analytical plates are usually spotted while preparative plates are streaked.

Step VIII: Spotting:

• Done with a melting point capillary tube or micropipette or microliter syringe.
• The applicator is charged by dipping the capillary end into the solution.
• The solution is then transferred by touching the tip of the capillary onto the adsorbent layer.
• The sample volume is usually in the range of 1-10l.
• The spot must be as small as possible for better separation and minimum tailing.

Step IX: Developing the chromatogram (Elution)

• Tank selection:
  • TLC can be developed in wide variety of chambers. For example, microscopic slides are developed in a small cylindrical glass jar or wide mouthed screw capped glass bottles.
  • Larger plates (20cm X 20cm) require a rectangular glass tank of suitable dimension with airtight lid.
• Tank saturation:
  • The inside of the development tank is lined with filter paper leaving a gap for viewing the chromaplate.
  • The filter paper is saturated with the selected developing solvent and the requisite amount of the developing solvent is carefully poured down the side of the tank.
  • Then the tank is closed with lid and allowed to stand for about 5 minutes so that the atmosphere in the tank becomes saturated with solvent vapour.
• Insertion of the plate:
  • The loaded plate with the origin spots or baseline towards the bottom of the tank is inserted and tilted.
  • Care should be taken that the solvent level is below the origin or baseline.
  • The tank is recapped or closed and the solvent is allowed to ascend by capillary action to the finishing line which is about 0.5mm away from the top edge of the adsorbent layer.
  • The time required to complete this development varies greatly with the solvent composition and the nature of the adsorbent.
  • When the solvent reaches the finishing line, the position of the solvent is marked on the absorbent layer and immediately the plate is removed from the development tank.
  • After removal the plate is dried in the fume cupboard.

Step X: Detection of spots/ Visualization of solutes:

• The position of colored components can be seen in daylight without any difficulty.
• For colorless solutes, visualization or detection techniques are many, such as:
• UV light:
  • Non-destructive method accomplished by viewing the plate under UV lamp at 254nm (short wavelength) and 350nm (long wavelength).
  • At short wavelength the plate is dark and compounds glow.
  • This method is limited to solutes capable of fluorescence.
  • At long wavelength, when a fluorescent indicator is incorporated into adsorbent the entire plate fluoresces green under UV light, and solutes appear as dark spot due to quenching or blocking effect of the fluorescent background.
• Iodine vapor:
  • This is a semi-destructive general method for most organic compounds.
  • The dried plate is allowed to stand in a closed tank containing iodine crystals scattered over the tank bottom.
  • The spots are revealed as brown stains.
  • Their positions should be marked as soon as the plate has been removed from iodine tank since standing in air for a short while causes the iodine to evaporate and the stained spots to disappear.

Thin layer Chromatography (TLC)-Principle and procedure