The Steps of TLC Analysis
Excellent discussions of sample preparation; sample application; mobile-phase selection; chromatogram development and documentation;
and zone detection, identification, and quantification in modern TLC are presented in two books (11,12), two book chapters
(13,14), and a review of TLC in drug analysis (15). This section contains brief discussions of some of these steps of the
TLC analytical process, followed by a more detailed description of stationary phases.
The most widely used methods for application of standard and sample solutions to a plate are manual with a capillary (for
example, 10- or 25-µL Drummond digital microdispenser) and automated instrumental application. Application of narrow, homogeneous
bands of controlled length has been shown to give superior chromatographic results compared to spot application. These automated
application devices are available from Camag and Desaga.
Choosing the Mobile Phase
Mobile phases are less polar than the silica gel layer (normal-phase TLC) and are usually composed of nonpolar and polar constituents
with or without an acid or base modifier to improve resolution. In reversed-phase TLC, the stationary phase is less polar
than the mobile phase, which often is a combination of methanol, acetonitrile, or tetrahydrofuran with water.
The mobile phase is optimized in terms of its strength and selectivity in relation to the sample and stationary phase. Snyder
classified solvents into eight groups within a selectivity triangle based on proton acceptor, proton donor, and dipole properties
(13). He later reviewed and expanded his studies of the role of the mobile phase in controlling selectivity for adsorption
TLC and suggested a seven-mobile-phase experimental design for optimizing relative retention and selectivity with binary solvent
mobile phases (16).
Plates are most often developed in a conventional glass large volume chamber (N-chamber) in the ascending direction. Also
available are twin trough chambers (TTCs), which allow the development of plates with a low volume of mobile phase and experimentation
with chamber saturation conditions, and an automatic developing chamber (ADC 2) that provides complete control of all aspects
of plate development (both from Camag).
Horizontal development chambers are also available from Chromdes and Camag (HDC 2). An advantage of these chambers is the
ability to develop the plate from both opposing sides towards the middle, permitting the number of samples to be doubled to
achieve even greater sample throughput.
Methods used to increase the resolution of complex mixtures include two-dimensional (2D) TLC, in which development of a single
sample applied to the corner of a single-layer plate is performed in perpendicular directions, with drying in between, using
two different mobile phases of about the same strength but complementary selectivities. A dual-layer plate (Multi-K, Whatman/GE
Healthcare) that allows both reversed-phase and normal-phase development is also available. The use of either of these can
spread sample components over the entire plate with high zone capacity. Figure 1 illustrates these two possibilities for 2D
Figure 1: 2D TLC on a single plate. Top TLC plate: a single layer (silica, C18, or cellulose) developed in two directions
with different mobile phases, 90° to one another, with drying between developments. Bottom TLC plate: a dual layer (C18 and
silica, adjacent to one another) developed in two different modes to improve separation of complex mixtures.
Note in each case, the components never need be removed from the plate before the second development is begun, saving time
and potential loss. After development, other procedures and documentation as described below can be continued.
The ease of application of 2D TLC is one example of an advantage of TLC compared to HPLC. It is much more difficult and costly
to accomplish a successful 2D HPLC method. 2D HPLC often requires the collection of sample fractions or complex column switching.
The biggest problem is often a mismatch of the mobile phase used in one mode compared to the second mode. If the mobile phases
are immiscible, then drying and reconstituting each sample fraction is usually required before introduction into the second
column. This is never a problem with 2D TLC because any initial mobile phase is evaporated from the plate before the second
development is started.
Multiple development either manually or with Camag's Automatic Multiple Development (AMD 2) instrument can also greatly improve
the resolution of complex mixtures. The typical mode of automatic multiple development uses a multiple-step mobile-phase gradient
with successive runs that have decreasing strength (less polar for a silica gel layer) and increasing development distance,
with vacuum drying of the plate between steps.
The development methods described above in this section involve migration of the mobile phase by capillary forces. There are
now devices available for techniques that are categorized as forced-flow planar liquid chromatography (FFPLC) with which the
mobile-phase migration is driven by other means. These include overpressured-layer chromatography (17), rotational planar
chromatography (18), and pressurized planar electrochromatography (19).