Thin-Layer Chromatography

84 3 Chemical Methods of Detection
Fig. 41: Automated dipping devices (DC Tauch-fix, BARON).
The advantage of such dipping apparatus is that the insertion and removal of the
chromatogram is performed at a uniform speed and the time of immersion can be
set as necessary. Interfering "ripple marks", such as are observed on manual
dipping, do not occur. Care must be taken, however, to clean off the back of the
Table 18. Statistical comparison (F-test [125]) of the methods. Standard deviation sXo of the
calibration curves for diethylstilbestrol and ethinylestradiol [114].
Procedure fra/w-diethylstilbestrol
sXo [ng per zone]
Manual dipping 0.37
Mechanical dipping 0.096
Statistical difference (F-test) significant
Ethinylestradiol
Sxo [ng per zone]
0.21
0.044
significant
3.2 Postchromatographic Detection 85
TLC plate which is wetted with reagent solution when it leaves the dipping bath
before laying it on the hotplate, laboratory bench or scanning stage.
The dipping solutions described in Part II of this book are usually less concentrated
than the corresponding spray solutions. The solvents employed are specially
chosen for their suitability to the special requirements of dipping solutions. Water,
which on the one hand, can sit on the surface of RP plates and not penetrate them
and, on the other hand, can cause disintegration of water-incompatible layers is
usually replaced by alcohol or other lipophilic solvents.
In general care should be taken in the choice of solvent to ensure that neither the
chromatographically separated substances nor their reaction products are soluble
in the solvent of the dipping reagent.
It is probable that the solvents given in the individual reagent monographs are not
suitable for all the substances with which the reagent will react. This point should
be taken into account especially for quantitative work and the user should make
appropriate modifications. In particular, there must be no loss of substance or
reaction product by dissolution (formation of "comet tails" by the chromatographic
zones).
When the plate is inspected the color intensity of the chromatogram zones must
be more intense at the top surface of the layer than it is when viewed from the
back of the TLC/HPTLC plate. If this is not the case the reagent must be made
less polar to avoid a frontal development across the thickness of the layer.
Fig. 42: Chromatogram of polycyclic aromatic hydrocarbons on caffeine-impregnated precoated
silica gel 60 HPTLC plates with concentrating zone (MERCK). The following can
be recognized in increasing Rf value. - 1. benzo(ghi)perylene, 2. indeno(l,2,3-cd)pyrene,
3 benzo(a)pyrene, 4. benzo(b)fluoranthene, 5. benzo(k)fluoranthene, 6. fluoranthene.