Page 124 - phytochemistry general program

Samples to be fractionated are applied as 0.1-1% solutions by means of
micropipettes or syringes or even glass capillaries (~ 1 mm outside diameter).
Usually 1-100 μl volume dissolved in a suitable volatile organic solvent is spotted
just by touching the layer, where the solution will be delivered. As many as 20
spots can be applied along one edge of a 20 X 20 cm plate, a spotting template is
usually used. The spot should be about 1-3 mm in diameter. In adsorption TLC,
the amount of sample per spot can be as high as 0.5 mg, but the load should not
exceed 0.05 mg when partition and other principles of chromatography are

employed.

Developing solvents (mobile phases) .2

The choice of the developing solvent is determined by the principle of
chromatography to be employed as well as the nature of samples.

Development

Chromatoplates are usually developed once by the ascending technique, at room
temperature, to a height of about 15-18 cm (or according to the dimensions of
plates). Jars and tanks of different sizes lined with filter papers are used to assure
saturation of the atmosphere with solvent vapors. This affects faster separation,
straight solvent front, and the formation of round spots, Lining with filter paper is

not necessary when the S-chamber or the BN-chamber are used.

Developments can be achieved at temperatures higher or lower than ambient
temperatures.

Types of developments
Several methods of development are used. The choice of any of them depends on

the experimental conditions and the sample as well.
(1) Single development, where the chromatoplate is developed once.

(2) Repeated chromatography, which can be:
a- Multiple development in the same solvent.

122