Lipid analysis：Thin layer chromatography

Lipid analysis

Thin layer chromatography is based on the separation of a mixture of compounds as it migrates with the help of a suitable solvent through a thin layer of adsorbent material which has been applied to an appropriate support. Thin layer chromatogram can be looked upon as being "open column" system. Following the development of a relatively simple apparatus for preparing chromatoplates with uniformly thin layers of adsorbent by Stahl (1964), TLC became very popular. Nowadays, ready-to-use TLC plates are normally used, since they offer greater reproducibility and higher mechanical strength.Different stationary phases are available that allow to separate by partition, ion-exchange, adsorption or a combination of any of these phenomena. Adsorption is the most common mechanism. In adsorption TLC the sample is continually fractionated as it migrates through the adsorbent layer. Competition for active adsorbent sites between materials to be separated and the developing solvent produces continuous fractionation. A portion of the material to be separated will be found in the mobile phase and a portion will be adsorbed to the solid adsorbent particles. As the process continues the various components move different distances, depending on their relative affinities for the adsorbent as compared with the migrating solvent. In general, the more polar compounds are held back by the adsorbent while less polar material advance further. Polar solvent effect the most movement of sample material over the adsorbent.

The migration of a compound in a given TLC system is described by its Rf value:

distance traveled by compound Rf = distance of the solvent front from the origin

i. Adsorbent:

The most common adsorbent used is Silica gel (silicic acid combined with a small amount of gypsum as a binding agent). Many other material have also proven useful for specific purposes; for example, alumina for the separation of steroids and water soluble vitamins and Kieselgur for separation of sugars.

ii. Solvents:

Although it is frequently possible to select a good solvent system by the empirical methods, some time and effort may be saved by consideration of some principles of the chromatographic separation. Of particular importance in TLC are the elutropic series of solvents. This is a series of solvents arranged in the order of their eluting power; that is having increasing ability to remove compounds from an adsorbent. A knowledge of the relative adsorbent characteristics of the class of compounds to be separated is also important. For example, saturated hydrocarbons are adsorbed poorly while unsaturated hydrocarbons are adsorbed according to the increase in number of their double bonds.

iii. Development:

The apparatus used for development must have certain features:

a) be capable of retaining any of a multitude of mixtures of original solvents

b) be air tight, to insure maintenance of a solvent saturated atmosphere

c) be relatively easy to handle

iv. Visualization:

Compounds separated can be visualized by

a) natural colour

b) spray reagents; yields coloured derivatives on reaction with the separated spots.

c) iodine vapour

d) fluorescence and UV absorption

e) autoradiography