Special considerations for identification and detection in Thin Layer Chromatography

Special considerations for identification and detection in Thin Layer Chromatography
Developed Chromatographic Sheet
Developed Chromatographic Sheet

Thin Layer chromatography presents a low cost, simple and sensitive option for identification and estimation of individual components in a mixture of components. The popular choices offered are Paper chromatography and HPTLC or High Performance Thin layer chromatography. Each of these techniques comprises essentially of a plane surface of paper or a metallic glass plate coated with an appropriate adsorbent with the help of a binder. The mobile phase drives the sample components by capillary action or gravitational force in case of suspended plates

Thin layer chromatography has found wide range of applications in drug development, organic chemistry research, foods, forensics, biochemical separations and research. Although the technique is simple and affords a visual display of separated spots the accuracy and precision is dependent on the technique adopted for application of sample spots, development and quantification of separated components.

Retardation Factor Rf

Retardation factor is an important parameter in thin layer determinations. It defines the distance travelled by the mixture component from application or starting point relative to the distance travelled by the solvent front over a given time period. Rf lies between 0 and 1.0. Ideally two spots should not overlap with each other.

Rf is influenced by impurities in the paper or solvent mixture, temperature and degree of saturation of the vapour inside the development chamber.

Choice of Solvent Mixtures

Judicious choice of solvent mixtures is critical and in thin layer separations. Special attention should be placed on the following aspects:

  • Solvents selected should be fully miscible with each other
  • Solvents should be chemically inert towards the sample mixture components
  • Any of the solvent component should not be volatile to an extent that can result in changes in the composition of sample mixture over the development time
  • The solvent should not result in distortion of spots through tailing

The minimum separation between two closely spaced spots should have Rf between 0.05 and 0.1

Detection of spots

In this stage the solvent front is marked and the solvent is dried. The commonly used practice is natural air drying or drying with a hair dryer to accelerate the drying. However, never hold the hair dryer too close to the plate so as to prevent local heating.

The technique for characterization and evaluation of spots include

  • Natural colour observation as in case of coloured compounds
  • Complexation reactions with colour producing reagents
  • Spraying for inducing fluorescence
  • The measurement of radioactivity

Iodine vapour is used for visualisation of colourless or non-fluorescent spots. Other commonly used reagents are :

Reagent Typical compounds
Ninhydrin Amino acids
2,4 – dinitrophenyl hydrazine Aldehydes&Ketones
Chloroplatinic acids Alkaloids
Bromothymol blue Lipids

Commercially available plates are available incorporate fluorescent dyes in the adsorbent. On holding the spotted plates in the UV viewing cabinet the spots glow and become visible due to fluorescence properties.

The two plates can be photographed before disposal to retain permanent record of separations as the plates have a tendency to fade with time.

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