Page 258 - Vogel's TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS

8 COLUMM AN0 THlN-lAVER LlilUlO CHROMATOCRAPHV

of separations and in quantitative measurements; these developments are
referred to as high-performance thin-layer chromatography (HPTLC). A brief
account of the technique of thin-layer chromatography follows together with a
summary of the main features of HPTLC (Section 8.7).
Technique of thin-layer chromatography. Preparation of the plate. In thin-layer
chromatography a variety of coating materials is available, but silica gel is most
frequently used. A slurry of the adsorbent (silica gel, cellulose powder, etc.) is
spread uniformly over the plate by means of one of the commercial forms of
spreader, the recommended thickness of adsorbent layer being 150-250 pm.
After air-drying overnight, or oven-drying at 80-90 OC for about 30 minutes,
it is ready for use.
Ready to use thin-layers (i.e. pre-coated plates or plastic sheets) are
commercially available; the chief advantage of plastic sheets is that they can be
cut to any size or shape required, but they have the disadvantage that they
bend in the chromatographic tank unless supported.
Two points of practical importance may be noted here:
1. care should be exercised in handling the plate to avoid placing fingers on
the active adsorbent surface and so introducing extraneous substances;
2. pre-washing of the plate is advisable in order to remove extraneous material
contained in the layer, and this may be done by running the development
solvent to the top of the plate.
Sample application. The origin line, to which the sample solution is applied, is
usually located 2-2.5 cm from the bottom of the plate. The accuracy and
precision with which the sample 'spots' are applied is, of course, very important
when quantitative analysis is required. Volumes of 1,2 or 5 pL are applied using
an appropriate measuring instrument, e.g. an Agla syringe or a Drummond
micropipette (the latter is a calibrated capillary tube fitted with a small rubber
teat). Care must be taken to avoid disturbing the surface of the adsorbent as
this causes distorted shapes of the spots on the subsequently developed
chromatogram and so hinders quantitative measurement. It may also be noted
that losses may occur if the usual method of drying applied spots in a gentle
current of air is used. The use of low boiling point solvents clearly aids drying
and also helps to ensure compact spots ( k2-3 mm diameter).
Development ofplates. The chromatogram is usually developed by the ascending
technique in which the plate is immersed in the developing solvent (redistilled
or chromatographic grade solvent should be used) to a depth of 0.5 cm. The
tank or chamber used is preferably lined with sheets of filter paper which dip
into the solvent in the base of the chamber; this ensures that the chamber is
saturated with solvent vapour (Fig. 8.6). Development is allowed to proceed
until the solvent front has travelled the required distance (usually 10-15 cm),
the plate is then removed from the chamber and the solvent front immediately
marked with a pointed object.
The plate is allowed to dry in a fume cupboard or in an oven; the drying
conditions should take into account the heat- and light-sensitivity of the
separated compounds.
The positions of the separated solutes can be located by various methods.
Coloured substances can be seen directly when viewed against the stationary
phase whilst colourless species may usually be detected by spraying the plate