Chromatogram Development

Thin-layer chromatography differs from all other chromatographic techniques in the fact that in addition to stationary and mobile phase a gas phase is present. This gas phase can significantly influence the result of the separation.

Processes in the Developing Chamber

The »classical« way of developing a chromatogram is to place the plate in a chamber, which contains a sufficient amount of developing solvent. The lower end of the plate should be immersed several millimeters. Driven by capillary action the developing solvent moves up the layer until the desired running distance is reached and chromatography is stopped. The following considerations primarily concern silica gel as stationary phase and developments, which can be described as adsorption chromatography.

Provided the chamber is closed, four partially competing processes occur:

1. Between the components of the developing solvent and their vapor, an equilibrium will be established eventually (1). This equilibrium is called chamber saturation. Depending on the vapor pressure of the individual components the composition of the gas phase can differ significantly from that of the developing solvent.
2. While still dry, the stationary phase adsorbs molecules from the gas phase. This process, adsorptive saturation, is also approaching an equilibrium in which the polar components will be withdrawn from the gas phase and loaded onto the surface of the stationary phase (2).
3. Simultaneously the part of the layer which is already wetted with mobile phase interacts with the gas phase. Thereby especially the less polar components of the liquid are released into in the gas phase (3). Unlike (1) this process is not as much governed by vapor pressure as by adsorption forces.
4. During migration, the components of the mobile phase can be separated by the stationary phase under certain conditions, causing the formation of secondary fronts.

Definition of plate and chamber formats

These format definitions are used in all CAMAG literature. Note: some plates can be developed in one direction only, e.g. plates with a concentration zone, GLP coded plates, etc. When you order plates make sure you understand the manufacturer‘s size definitions.

 In connection with the development process, the following aspects should be considered:

With the exception of single component liquids (neat solvents), developing solvent and mobile phase are, strictly speaking, not the same. Their composition changes with progressing chromatography. Unfortunately the terms »developing solvent« and »mobile phase« are often used as synonyms. In the true sense only the liquid in the chamber should be called developing solvent, while the liquid moving through the layer constitutes the mobile phase. Only the composition of the developing solvent at the time when it is placed into the chamber is positively known. The processes (1) and (2) can be experimentally affected by:

• Fitting the chamber more or less completely with filter paper soaked with developing solvent.
• Waiting a certain time between the introduction of developing solvent into the chamber and beginning of chromatography – chamber saturation.
• Allowing the plate to interact with the gas phase prior to chromatographic development, i.e. without contact to the developing solvent – preconditioning.

An interaction according to (2) and (3) can be effectively prevented by placing a counter plate at a distance of one or a few millimeters to the chromatographic layer. This is called »sandwich configuration«. The further an equilibrium according to (1) and/or (2) has been established and the less different the components of the mobile phase are in respect to their adsorption behavior, the less pronounced is the formation of secondary fronts resulting from (4). In well-saturated chambers and on preconditioned layers secondary fronts are often not observed. In sandwich configuration and particularly in OPLC secondary fronts are very prominent.

During chromatography, components of the developing solvent, which have been loaded onto the dry layer via the gas phase according to (2), are pushed ahead of the true but invisible solvent front. Exceptions are very polar components such as water, methanol, acids, or bases. This results in Rf values being lower in saturated chambers and particularly on pre-conditioned layers, than in unsaturated chambers and sandwich configurations.

Reproducible chromatogram development, here several plates under UV366 nm. Influence of the activity of the layer (relative humidity) on the separation at equal migration distance. From left: rH 18%, 47% and 75% The desired activity is set in only a few minutes

Thin-layer Chromatography in most cases proceeds in a non-equilibrium between stationary, mobile, and gas phase. For this reason it is very difficult to correctly describe the conditions in a developing chamber.
Reproducible chromatographic results can only be expected when all parameters are kept as constant as possible. Chamber shape and saturation are playing a predominant role in this regard. Unfortunately this means that the chromatographic result is different in each chamber!
There are neither »good« nor »poor« chambers! However, in some chambers the parameters can be better controlled, i.e. reproduced, than in others.

Choosing a developing chamber

Selection of the »proper« chamber is done during method development and generally follows »practical« considerations such as which chamber is available, which one must be used due to an SOP, or which one has been used in the past if a results comparison is to be made. However, a focus should also be on economical aspects such as time requirement and solvent consumption. A selection of glass chambers can be found here.
The Horizontal Developing Chambers have proven to be exceptionally economical, flexible and reproducible in operation. Although designed for applications where the plate is developed from two sides, they are also suitable for single-sided developments in unsaturated, saturated and sandwich configuration as well as for preconditioning of HPTLC plates.
The new Automatic Developing Chamber (ADC 2) is unsurpassed for reproducibility and universal applicability. This instrument does not only eliminate any effects of the operator when introducing the plate into a saturated chamber, but also the activity of the layer prior to start of chromatography can be set and drying of the chromatographed plate is rapid and complete. For development a conventional 20 x 10 cm Twin Trough Chamber is used. This way chamber geometry and chromatographic conditions of already existing analytical procedures can be retained, but environmental and operational effects are standardized.
In case the sample contains polar and non-polar components, which must be separated in the same analysis, the principle of Automated Multiple Development (AMD) can be employed. Development is performed on the basis of a solvent gradient from polar to non-polar over several steps with intermediate drying.