Thin-Layer Chromatography (TLC) Development |
Overview of TLC Development Methods and Developing Chambers
TLC Plate-Development Methods
The plate-development is the main process of thin-layer chromatography in which the actual separation takes place. In this step, a solvent system is introduced to the sample on the TLC layer in order to separate the mixture into individual substances. Separation can be achieved through various methods. The most common TLC development techniques are explained below.
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Vertical Development
In this common TLC development method, the plate is placed in a suitable TLC developing chamber such that the solvent wets the TLC layer below the starting line. Due to capillary forces, the solvent rises up the layer, transporting the sample mixture. Once the solvent front has reached the predetermined height (10-15 cm for TLC and 3-7 cm for HPTLC), the plate is removed from the chamber, the solvent front is marked with a pencil or spatula, and the plate is dried.
Horizontal Development
In this TLC developing method, the plate is positioned horizontally inside the chamber, and the solvent is applied using a wick or capillary slit. Development can be performed from one or both sides of the TLC plate.
Two-Dimensional Development
In two-dimensional TLC development, the sample is applied to a starting point in a corner of the TLC plate. The plate is placed in a normal chamber and developed once from bottom to top. After drying, the plate is turned 90° and placed in another chamber with a different solvent and developed again. The chromatogram track from the first development is used as the starting line for the second development. Two-dimensional TLC offers the advantage of running a standard with either development. However, the standard cannot be developed in two dimensions on the same chromatogram since it would mix with the sample.
Multiple Development
In this method, the TLC plate undergoes multiple developments with drying between each cycle. The solvent travels repeatedly through the layer, re-concentrating and deforming the spots, often producing elliptical shapes or narrow bands. This significantly improves resolution for substances with Rf values below 0.5. Multiple development can be performed over different separation distances, using the same solvent or different solvents of varying polarity.
AMD (Automated Multiple Development)
AMD is a automated version of multiple development, and is based on a gradient solvent system. In the case of silica gel TLC layers, solvents of decreasing polarity are used, and each consecutive development step is performed over a longer distance. Ideally, development is performed in special U-chambers, where the solvent is fed via a pump. After each run, the solvent is drawn off under vacuum. Since the process starts with polar solvents, all substances first migrate with the front and become concentrated to narrow bands. As solvent polarity decreases, first the most polar and later the non-polar substances remain in position. AMD offers excellent resolution and sensitivity, and allows each step to be preselected at will.
TLC Developing Chambers
Thin-layer chromatography is usually performed in developing chambers. As explained below, there are a variety of chambers to choose from depending on the sample and application goals. Furthermore, TLC development can be performed in saturated or unsaturated chambers.
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Normal Chambers
Normal development chambers are the most common type used in thin-layer chromatography. These glass chambers measure 21 x 21 x 9 cm, and are suitable for simultaneous development of at most two TLC plates.
Twin-Trough Chambers
The twin-trough chamber is divided into two sections by a ridge at the base. For standard linear development, solvent consumption (20 ml for a 20 x 20 cm plate and 4 ml for a 10 x 10 cm plate) is considerably lower in twin-trough chambers than in normal chambers. This reduces material and disposal costs.
Sandwich Chambers (S-Chambers)
In this method, a sandwich is created by covering the TLC layer with a glass plate. The TLC layer and cover plate should be slightly separated using spacers such that only the bottom-most zone (about 2 cm in width) remains uncovered. The cover plate should not be dipped into the solvent. The sandwich design can be used in any type of TLC development chamber.
Horizontal Chambers (Linear Chambers)
In this method, a sandwich chamber is used for horizontal development of TLC plates. TLC development can be performed from one or both sides of the plate.