THIN LAYER CHROMATOGRAPHY (TLC)

 

•       Thin-layer chromatography (TLC), first described in 1938

•       Replaced paper chromatography 

•       Because of its rapid, more sensitive, and more reproducible. 

•       The resolution in TLC is greater than in paper chromatography because the particles on the plate are smaller and more regular than paper fibres

•       Separation of low molecular weight compounds.

•       Qualitative and quantitative sample analysis

•       Adsorption and partition chromatography

•       Adsorption – kieselguhr or alumina

•       Partition – silica gel

•       Kieselguhr is a natural diatomaceous earth that can be used for the separation of polar or moderately polar compounds.

•        Ideal chromatographic conditions for special applications, such as the separation of inorganic ions, herbicides, or steroids

Preparation and procedure

•       Slurry of stationary phase is made using solvent

•       Slurry poured onto glass plate and spread evenly.

•       Plates dried in oven at 40^oC.

•       Sample applied using micropipette and dried.

•       As in paper chromatography, the sample (in carrier solvent) is applied as a spot or streak about 1.5 cm from one end of the plate.

•       After evaporation of the carrier solvent, the TLC plate is placed in a closed developing chamber

•       Solvent migrates up the plate (ascending development) by capillary action, and sample components are separated.

•       Allow solvent to reach the top and dry the plate

•       Spray natural colours or reagents

•       iodine vapors, sulphuric acid, cerium sulphate

•       Spots can be seen 

•       retention factor (Rf) of each component is assessed

•       Rf= Distance travelled by amino acid

                            Distance travelled by solvent

Advantages

·       High sample throughput

·       Separations of complex mixtures

·       Low cost

·       Analysis of several samples and standards simultaneously

·       Minimal sample preparation

·       Possibility to store the plate for later identification and quantification.

• Short time required to perform the analysis (typically 5-10 min) 
• High sensitivity, with only a few micrograms (10^-6 g) of sample needed.

Applications

•       Industries and research fields

•       Including pharmaceuticals,

•       clinical testing,

•       environmental toxicology,

•       food, water and pesticide analysis, and

•       cosmetics

•       Aminoacids, nucleic acids, lipids, steroids, terpenoids, hydrocarbons, carbohydrates. 

Thin-layer chromatography sorbents and mode of separation

Sorbent	Chromatographic mechanism	Typical application
Silica gel	Adsorption	Steroids, amino acids, alcohols, hydrocarbons, lipids, aflatoxins, bile acids, vitamins, alkaloids
Silica gel RP	Reversed phase	Fatty acids, vitamins, steroids, hormones, carotenoids
Cellulose, kieselguhr	Partition	Carbohydrates, sugars, alcohols, amino acids, carboxylic acids, fatty acids
Aluminum oxide	Adsorption	Amines, alcohols, steroids, lipids, aflatoxins, bile acids, vitamins, alkaloids
polyethyleneimine cellulose	Ion exchange	Nucleic acids, nucleotides, nucleosides, purines, pyrimidines
Magnesium silicate	Adsorption	Steroids, pesticides, lipids, alkaloids