Page 198 - Multidimensional Chromatography

Multidimensional Planar Chromatography 191

lower plate enables on-line detection of the compounds after ‘anticircular’ develop-
ment. Off-line detection can be used when circular development is complete.
Although this combination might seem complicated, MD-PC, partly in combination
with multiple development, can easily be realized with little modification of com-
mercially available equipment (37).

8.13 COMBINATION OF MULTIDIMENSIONAL
PLANAR CHROMATOGRAPHIC METHODS

In the introduction to this chapter, MD-PC was defined as a procedure in which sub-
stances to be separated were subjected to at least two separation steps with different
mechanisms of retention (5). Discussion of the basic potential modes of operation
showed that because of the versatility which resulted from being able to combine
mobile phases of different composition, more than two development steps can easily
n
be realized by the use of D techniques.
Working with the same mobile phase composition, the serial connection of sta-
tionary phases under capillary-controlled flow conditions is much more complicated,
for two reasons. First, for HPTLC plates the mobile phase velocity might no longer
be adequate to maintain optimum separation conditions, and secondly, the handling
of more than three chromatographic plates within a separation distance of 8 cm is
practically impossible. Unfortunately, only one type of bilayer (silica and RP-18) is
commercially available. However, 10 cm 20 cm glass-backed plates coated with
chemically modified layers are available and can be cut to (3–5) cm 20 cm, thus
enabling many stationary-phase combinations to be realized by the graft technique.
The best technique is, therefore, a parallel combination of stationary and mobile
phases. In order to take advantage of the double effect of MD-PC it is recommended
that bilayer plates are combined with multiple development techniques (38) in which
total solvent strength and mobile phase selectivity are changed simultaneously. By
the use of this technique, in the first direction S T and S V are varied in ‘n’re-chromato-
graphic steps (Figure 8.20(a)) and in the perpendicular, second, direction (Figure
8.20(b)) S T and S V are again varied in ‘m’ re-chromatographic steps (Figure 8.20(c)).
This is an extended version of MD-PC and can be regarded as being ‘double’ MD-
PC, because changes not only of the stationary phases (bilayer) but also of the
mobile phase composition ensure the criteria of multidimensional planar chromatog-
raphy. As is apparent from Figure 8.20(c), the number of compounds separated is
clearly more than for any other version.
n
The versatility of combining stationary phases, D, and forced-flow techniques is
illustrated in Figure 8.21. Separation in the first dimension is performed by applica-
tion of the first stationary and mobile phase combination (S T1 ; S V1 : S Tn ; S Vn ) by use
of multiple development. After drying of the chromatographic plate, the separation
can be continued in a perpendicular direction by using three prepared chromato-
graphic plates, with different characteristics (decreasing polarity), in combination
with a second mobile phase (S Tx ; S Vx ). It must be pointed out that the separating