Thin-Layer Chromatography

56 3 Chemical Methods of Detection
which increase the selectivity of the separation, increase the sensitivity of detection
and improve the linearity [4]. Trace analyses often only become possible after
chemical reaction of the substance to be detected. The aim of prechromatographic
derivatization is, thus, rather different than that of postchromatographic
derivatization, where the aim is first to detect the substance and then only secondarily
to characterize it (Fig. 30).
3.1 In Situ Prechromatographic Derivatization
There has for some years been a considerable backlog in the development of
practicable prechromatographic methods [5]. It is becoming more and more recognized
that the future direction to be taken by trace analysts is to make improvements
in the extraction, enrichment and clean-up of the sample and in the optimization
of derivatization. It is only in this way that it is possible to employ the sensitive
chromatographic techniques optimally for the solution of practically relevant
problems.
About 100 000 new chemical compounds are synthesized every year [6]; these have
to be recognized, identified and determined quantitatively. Ever more frequently
this is only possible because of the employment of multiple chromatographic
methods coupled with derivatization during or before the separation process.
For these reasons "Reaktions-Chromatographie" [7] ("Chromatographie fonctionelle
sur couche mince" [1,2]) is steadily gaining in importance. Here the reaction,
which also then takes on the role of a clean-up step, is performed at the start or
in the concentration zone of the TLC plate.
The requirements of such a reaction are [8]:
• single, stable reaction products,
• high yields in all concentration ranges,
• simplicity and rapidity in application,
• no interference by excess reagent with the chromatographic separation and
analysis that follows.
Such in situ reactions are based on the work of MILLER and KIRCHNER [9] and
offer the following possibilities [10]:
• The reaction conditions can be selected so as to be able to separate substances
with the same or similar chromatographic properties (critical substance pairs)
by exploiting their differing chemical behavior, thus, making it easier to identify
them. Specific chemical derivatization allows, for example, the esterification of
primary and secondary alcohols which can then be separated from tertiary
alcohols by a subsequent chromatographic development (Sec. 3.1.5). It is just
as simply possible to separate aldehydes and ketones produced by oxidation at
the start from unreactive tertiary alcohols and to detect them group-specifically.
• The stability of the compound sought (e.g. oxidation-sensitive substances) can
be improved.
• The reactivity of substances (e.g. towards the stationary phase) can be reduced.
• The stability of the compound sought (e.g. oxidation-sensitive substances) can
be improved.
This, on the one hand, reduces the detection limit so that less sample has to be
applied and, thus, the amounts of interfering substances are reduced. On the other
hand, the linearity of the calibration curves can also be increased and, hence, fewer
standards need to be applied and scanned in routine quantitative investigations
so that more tracks are made available for sample separations. However, the
introduction of a large molecular group can lead to the "equalization" of the
chromatographic properties.
In practice, reaction chromatography is usually performed by first applying spots
or a band of the reagent to the start zone. The sample is usually then applied while
the reagent zone is still moist. Care should be taken to ensure that the sample
solvent does not chromatograph the reagent outwards. The reagent solution can
be applied once more, if necessary, to ensure that it is present in excess. There is
no problem doing this if it is employed as a band with the Linomat IV, (Fig. 31],
for instance.
If heat is necessary to accelerate the reaction, the start zone should be covered by
a glass strip before being placed on a hotplate or in a drying cupboard. After
reaction is complete the TLC plate should be dried and development can begin.
There have also been repeated descriptions of coupling in the sense of a twodimensional
S —R—S (separation — reaction — separation) technique. In this
case the chromatogram track from the first separation serves as the start zone
for a second chromatographic development after turning the plate at 90°. The
derivatization described above is performed between the two chromatographic
separation steps.
Reactions can also occur during chromatographic development. These can either
be undesired reactions or planned derivatizations. Thus, WEICKER and BROSSMER
[11] have reported, for example, that hexoses, pentoses and disaccharides can be
aminated when ammonia-containing mobile phases are employed on silica gel G
layers. On the other hand, fluorescamine or ninhydrin have been added to the