Page 186 - Multidimensional Chromatography

Multidimensional Planar Chromatography 179

(a) (b) (c)
n n n
25 25 25

3 3 3

2 2 2

1 1 1

Figure 8.7 Schematic diagram of the zone refocusing mechanism: (a) the applied sample
before starting the separation; (b) the solvent front reaches the first stage (n 1), where the
sample starts to refocus; (c) the solvent front reaches the second stage (n 2), where all com-
pounds together start to migrate.

molecules still ahead of the solvent front (10). When the solvent front moves beyond
the front edge of the zone, the refocused zone starts to migrate and is broadened by
diffusion in the normal way. By use of optimum conditions a balance between zone
refocusing and zone broadening can be achieved.
A detailed description of the versatility of multiple development techniques in
one dimension has been given by Szabady and Nyiredy (18). These authors com-
pared conventional TLC with unidimensional (UMD) and incremental (IMD) multi-
ple development methods by chromatographing furocoumarin isomers on silica
using chloroform as the monocomponent mobile phase. The development distance
for all three methods was 70 mm, while the number of development steps for both of
n
the D techniques was five. Comparison of the effects of UMD and IMD on zone-
centre separation and on chromatographic zone width reveals that UMD increases
zone-centre separation more effectively in the lower R f range, while IMD results in
narrower spots (Figure 8.8).
For the analysis of multicomponent mixtures spanning a wide polarity range, the
simplest form of stepwise gradient development (GMD) is required (18). Separation
n
of components into fractions of increasing polarity can be achieved by D with the
same chromatographic development distance and a mobile phase gradient of increas-
ing solvent strength. In the first chromatographic step the full length of the layer is
developed with the weakest solvent system, optimized for the separation of non-polar
compounds. Semi-polar and polar compounds do not migrate with this mobile phase.