Page 130 - phytochemistry general program

(1) Thick layers of adsorbents are usually used (20X20 cm, 0.5-2 mm thick). They
are prepared by pouring and are allowed to air-dry for several hours before

activation in an oven to prevent cracking.

(2) A concentrated solution of the sample is applied to the chromatoplate as a
band, either manually, with a simple syringe or with a“ streak applicator”.

(3) Multiple developments is usually recommended to improve separation.

(4) After development, bands are detected by non-destructive methods (or
destructively using edge spray technique or pilot spots.

(5) The adsorbent is scraped off with a razor blade or aspirated with a zone
extractor.

(6) The desired material is eluted by several portions of a suitable solvent and is
recovered from the solution after filtration through a sintered glass funnel and the

solvent is evaporated.

(7) Fractions that are not completely resolved can be re-chromatographed again

Quantitative TLC Analysis .3

Thin-layer chromatography, particularly adsorption, has been used for quantitative
analysis. Fractions or zones can be determined either directly on the plate or after

elution with a suitable solvent(s) and evaporation.

A- Quantitative TLC on the plate

Spot Area Method .1

The size of a spot generally increases with the amount of material it contains,
but not in a linear fashion. The areas of the spots in the sample are compared
with those of a series of standards, which are used for plotting a calibration

curve.

Photodensitometry .2

Densitometers based on measuring transmittance, reflectance and
fluorescence or fluorescence quenching are sometimes used. e.g. Zeiss TLC

spectrophotometer.

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