Development chambers
The development process in TLC can be carried out in a variety of vessels that differ significantly in design and sophistication. For convenience these are often categorised under the headings of normal (N-chamber) and sandwich (S-chamber), and further subdivided according to whether the internal atmosphere is saturated (NS or SS) or unsaturated (NU or SU). Sandwich chambers have a depth of gas phase in front of the layer of less than 3 mm, with other chamber designs indica ted as normal chambers. Saturation of the vapour phase is achieved by using solvent-saturated pads or filter papers as a chamber lining. The twin-trough chamber is the most popular of the simplest TLC developing chambers. It consists of a standard rectangular developing tank with a raised, wedge-shaped bottom. The wedged bottom divides the tank into two compartments, so that it is possible either to develop two plates simultaneously or to use one compartment to condition the layer prior to development (Fig. 1). The horizontal developing chamber (Fig.2) can be used in either the normal or sandwich configuration for either conventional edge-to edge or simultaneous edge-to-centre develop ment. Starting the development simultaneously from opposite edges allows the number of samples separated to be doubled in the same time. The sandwich configuration of the horizontal developing chamber is not suitable for mobile phases that contain volatile acids, bases or large amounts of volatile polar solvents, such as methanol or acetonitrile, because of the restricted access of the saturated vapour phase to the dry portion of the separation layer.
Figure 1 Diagram showing pre-equilibration of plate and development in twin trough tank.
The automated developing chamber increases laboratory productivity and improves the reproducibility of separations by providing precise control of layer conditioning, mobile-phase composition, solvent-front migration distance and drying conditions. This chamber can be used in the normal or sandwich configuration with all the operational features preselected on a microprocessor-based control unit and monitored by sensor technology. The AMD chamber (Fig. 3) provides the necessary conditions and control for automated separations by incremental multiple develop ment with a decreasing solvent-strength gradient. The operating parameters of layer conditioning, solvent-front migration distance, mobile-phase composition and drying time for each development, and the total number of developments for the separation, are entered into the computer-based control unit. The complete separation sequence is carried out without further intervention. Each development is typically 3–5 mm longer than the previous one and, depending on the complexity of the desired mobile phase gradient, a total of 10–30 develop ments is used, which requires 1.5–4.5 h for completion.
Figure 2 Illustration of a horizontal developing chamber: 1, TLC plate; 2, removable counter plate to convert the chamber from normal to sandwich configuration; 3, mobile-phase reservoirs; 4, glass slide to direct mobile phase flow; 5, glass cover plate. Raising the glass slide (4) to contact the edge of the layer (1) causes the mobile phase to flow through the layer from the solvent reservoir (3). If both glass slides are raised simultaneously, development occurs from both sides to the middle, which allows the simultaneous development of samples applied along the two edges of the layer.
Figure 3 Illustration of the automated multiple-development chamber: 1, developing chamber; 2, solvent reservoirs; 3, solvent-selection valve; 4, solvent mixer; 5, wash bottle for preparation of the gas phase for layer conditioning; 6, gas phase reservoir; 7, vacuum pump; 8, solvent waste reservoir. All operations of this chamber are managed by a programmable control unit and initiated as time sequences. A sequence starts with a conditioning step in which the vapour phase stored in reservoir (5) is pumped into the developing chamber (1). During conditioning the mobile-phase composition is mixed by withdrawing the desired solvents from their storage bottles (2) and transferring the desired volumes to the mixer. At the selected time the mobile phase is pumped into the developing chamber and ascends the layer to the desired height, determined by an optical position monitor. The solvent is then drained from the chamber to waste (8) and the vacuum pump (7) engaged to remove solvent vapours from the chamber. The next segment of the sequence is then commenced according to the program entered into the control unit, until all the segments are complete and the separation is finished.
