Development
The principal development techniques in TLC are linear, circular and anticircular, with the velocity of the mobile phase controlled by capillary forces or forced-flow conditions. Circular TLC typically employs centrifugal force in the separation. The sample is applied as a band at the centre of a circular plate. The plate is then spun in a centrifugal manner, with the mobile phase in contact with the centre of the plate. The centrifugal force causes the mobile phase to move radially across the plate to the outer edge, with separation of the components of the sample taking place in the process. The separation process can either be interrupted and the separate bands developed or the bands can be allowed to develop right to the edge of the plate and then be collected for further examination. Anticircular TLC operates in a reverse manner, with the sample and mobile phase applied at the outer edge of a circle.
In any of these modes, continuous or multiple development can be used to extend the application range. Radial development is used rarely for drug analysis. Forced-flow development requires sophisticated equipment not commonly found in analytical laboratories, and is not described here. For linear (or normal) development, samples are applied along one edge of the plate and the separation developed for a fixed distance in the direction of the opposite edge. Viewed in the direction of development, the chromatogram consists of a series of compact symmetrical spots of increasing diameter or, if samples are applied as bands, in rectangular zones of increasing width (Fig. 1).
In continuous development, the mobile phase is allowed to traverse the layer under the influence of capillary forces until it reaches some predetermined position on the plate, at which point it is evaporated continuously. Evaporation of the mobile phase usually occurs at the plate atmospheric boundary using either natural or forced evaporation. Continuous development is used primarily to separate simple mixtures with a short development length and a weaker (more selective solvent) than employed for normal development.
Figure 1 Development of TLC plate with (A) sample applied to the plate as a spot and (B) sample applied to the plate as a band.
In unidimensional multiple development, the TLC plate is developed for some selected distance, then either the layer or the mobile phase is withdrawn from the developing chamber, and adsorbed solvent is evaporated from the layer before repeating the development process. The principal methods of unidimensional multiple development are summarised in Table 13.7. Multiple development provides a very versatile strategy for separating complex mixtures, since the primary experimental variables of development distance and composition of the mobile phase can be changed at any devel opment step, and the number of steps can be varied to obtain the desired separation. Multiple development provides a higher resolution of complex mixtures than does normal or continuous development; it can easily handle samples of a wide polarity range (stepwise gradient devel opment); and, because the separated zones are usually more compact, it leads to lower detection limits. Equipment for automated multiple development is commercially available. For drug mixtures that span a wide retention range, some form of gradient development is required to separate all the components in either a single chromatogram or in separate chromatograms for successive developments. Continuous solvent-composition gradients, as commonly employed in HPLC, are used rarely in TLC. These require experimental conditions that Are less convenient than those for mobile-phase step gradients. In addition, step gradients can be constructed easily to mimic a continuous linear gradient, with the added advantage that the zone refocusing effect can be employed to minimise zone broadening. Gradients of increasing solvent strength are used to fractionate complex mixtures by separating just a few components in each step.
Individual drugs are usually identified and quantified at the intermediate step sat which the drugs of interest are separated. In this way, the zone capacity can be made much larger than predicted for a complete separation recorded as a single chromatogram. However, this approach is
Tedious when many components are of interest and it is difficult to automate. Alternatively, if incremental multiple development is used, the sample can be separated for the shortest distance in the strongest mobile phase, with each subsequent,
Longer development using mobile phases of decreasing solvent strength. This strategy is most useful when the final separation is to be recorded as a single chromatogram,
but it is limited in zone capacity because all the components must be located between the sample origin and the final solvent front. The two approaches for exploiting solvent-strength gradients are thus complementary and selection is made based on the properties of the sample.
The decreasing solvent-strength gradient approach is the operating basis of automated multiple-development (AMD)chambers.
In two-dimensional TLC the sample is spotted at the corner of the layer and developed along one edge of the plate. The solvent is then evaporated, and the plate is rotated through 90 and redeveloped in the orthogonal direction. If the same solvent is used for both developments, the sample is redistributed along a line from the corner at which the plate was spotted to the corner diagonally opposite (see Fig. 2). In this case, only a small increase in resolution can be anticipated. For a more efficient separation system, the resolved sample should be distributed over the entire plate surface. This can be achieved only if the selectivity of the separation mechanism is complementary in the orthogonal directions. Using two solvent systems with complementary selectivity is the simplest approach for this, but it is often only partially successful. In many cases the two solvent systems differ only in their intensity for a given set of properties and are not truly orthogonal. Chemically bonded layers can be used in the reversed-phase and normal-phase modes, and these enable the use of additives and buffers as a further way to adjust selectivity. The acceptance of two-dimensional TLC for quantitative analysis will depend, though, on providing a convenient method for in-situ detection and data analysis. It seems unlikely that two-dimensional develop ment will be more widely used in TLC, except for qualitative analysis, until the problems of detection are solved.
Figure 2 Two-dimensional TLC as illustrated by the separation of lipid compounds. (Adapted from Parsons and Patton 1967.)
