Page 259 - Vogel's TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS

THlN-lAVER CHROMATOCRAPHV 8.6

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Knob-
Lid ---

Plate in the-

Fig. 86 Reproduced from D. Abbott and R. S. Andrews, An Zntroakction to Chromatography,
Longman, London, 1965.

with an appropriate* reagent which produces coloured areas in the regions which
they occupy. Some compounds fluoresce in ultraviolet light and may be located
in this way. Alternatively if fluorescing material is incorporated in the adsorbent
the solute can be observed as a dark spot on a fluorescent background when
viewed under ultraviolet light. (When locating zones by this method the eyes
should be protected by wearing special protective goggles or spectacles.) The
spots located by this method can be delineated by marking with a needle.
Quantitative evaluation. Methods for the quantitative measurement of separated
solutes on a thin-layer chromatogram can be divided into two categories. In
the more generally used in-situ methods, quantitation is based on measurement
of the photodensity of the spots directly on the thin-layer plate, preferably using
a densitometer. The latter instrument scans the individual spots by either
reflectance or absorption of a light beam, the scan usually being along the line
of development of the plate. The difference in intensity of the reflected (or
transmitted) light between the adsorbent and the solute spots is observed as a
series of peaks plotted by a chart recorder. The areas of the peaks correspond
to the quantities of the substances in the various spots. This type of procedure
requires comparison with spots obtained using known amounts of standard
mixtures which must be chromatographed on the same plate as the sample. The
design and specifications of commercially available densitometers have been
re~iewed.~~
The alternative, and cheaper, procedure is to remove the separated components
by scraping off the relevant portion of the adsorbent after visualisation by a
non-destructive technique. The component is conveniently extracted by placing
the adsorbent in a centrifuge tube and adding a suitable solvent to dissolve the
solute. When the solute has dissolved the tube is spun in a centrifuge, the
supernatant liquid removed and analysed by an appropriate quantitative
technique, e.g. ultraviolet, visible or fluorescence spectrometry or gas-liquid
chromatography. Alternatively the solute may be extracted by transferring the
adsorbent on to a short column of silica gel supported by a sinter filter and
eluting with the solvent. Again the extract is analysed by a suitable quantitative
technique. In each case, of course, it is necessary to obtain a calibration curve

* Spraying of locating reagents should always be performed in a fume cupboard.