THEORY AND PRINCIPLE
The major features controlling the resolution of the TLC plate are “spot”
size and the physical dimension of the plate. Normally if a spot diameter is 0.5
cm. and a plate length of 10 cm. then optimally only 20 analytes could be
completely resolved. In chromatography the distribution coefficient (K) of a
component is defined by the relation:
Where csp is the concentration of the component in unit volumes of the
stationary phase, and Cmp is the concentration of the component in unit volume
of the mobile phase. K describes the relative affinity of a component for the two
phases and hence relates to the distance and speed with which it moves through
the plate on elution. Distribution coefficients and rates of migration , however are
difficult to evaluate. In TLC and paper chromatography the results obtained are
described by quoting the Rf values, which refers to migration “relative to the
solvent from“ and was shown as
Rf = Distance traveled by solute
Distance travelled by solvent front
OUTLINE OF THE METHOD
In thin layer chromatography a solution of the sample in a volatile solvent
is applied via glass capillary tubes approximately 1-2 cm. from the bottom of a
uniform layer of inert adsorbent, such as silica gel or alumina, which has been
uniformly spread over a suitable supporting plate such as glass or plastic and
dried under standard conditions. When the spot has been dried the plate is placed
vertically in a suitable tank with its lower edge immersed in the selected mobile
phase. The solvent rises by capillary action and an ascending chromatographic.
separation is thus obtained , resolving the sample mixture into discrete spots.
At the end of the run, the solvent is allowed to evaporate from the plate and the
separated spots are located and identified either by physical methods such as
visual inspection, fluorescence or radiation monitoring or by chemically reacting
with developing reagent , the method chosen is dictated by the composition of the
In thin layer chromatography only small amounts of adsorbent and minute
samples are needed. The separated spots are located on the plate using a variety of
visualization techniques in common with paper chromatography. Afier separation,
it is easy to recover an individual substance by scraping off and collecting the part
of the layer on which the spot is adsorbed. The substance can then be extracted
with a suitable solvent.
The general properties of adsorbent for thin layer chromatography should
be of two types
1. Particle size
2. Homogeneity of the absorbent.
A particle size of l- 25 pm is usually recommended. A coarse grained
material will not produce a satisfactory thin layer. It is also visualized that strong
adsorbed substances require strong eluents , and weakly adsorbed materials
weaker eluents. It is helpful to know which features of chemical constitution
affect the strength of the adsorption band. Some examples of adsorbents ,which
have been used for representative separations by thin layer chromatography , are
given as .
BASlC OPERATIONS INVOLVED
The basic operations which are involved in TLC are the following:
1. Preparation of chromatoplates.
2. Application of samples to ehromatoplates.
3. Choice of adsorbent.
4. Selection of solvent
5. Proper developing system.
6. Location of compounds on chromatoplates.
7. Detection and Identification.