Thin layer chromatography
TLC is a widely used technique for the separation and identification of drugs. It is equally applicable to drugs in their pure state, to those extracted from pharmaceutical formulations, to illicitly manufactured materials, and to biological samples.
In its simplest form, TLC can be regarded as the separation of substances on a planar surface coated with a stationary phase. The mobile phase moves across the planar surface from one edge to the other and substances separate out according to their distribution between the stationary phase and mobile phase (seeFig.1). As we will see in the discussion that follows, this is a simple view of TLC and many adaptations and refinements have been made to this basic procedure to enable rather sophisticated separations.
TLC as we know it today was established in the 1950s (see Fig. 2) with the introduction of standardised procedures that led to improved separation performance and reproducibility, and paved the way for its commercialization and an increase in the number of published applications. The 1970s saw the introduction of fine particle layers and associated instrumentation required for their correct use. In this form, TLC became known as high-performance TLC, instrumental TLC or modern TLC to distinguish it from its parent, now generally referred to as conventional TLC. High-performance TLC has not displaced conventional TLC from laboratory studies and the two approaches coexist today because of their complementary features (Table 2). Conventional TLC provides a quick, inexpensive and portable method for qualitative analysis. It requires minimal and readily available instrumentation and uses easily learned experimental techniques. HPTLC is characterised by the use of kinetically optimised layers for faster and more efficient separations; it takes advantage of a wider range of sorbent chemistries to optimise selectivity and requires the use of instrumentation for convenient (auto mated) sample application, development and detection. HPTLC provides accurate and precise quantitative results based on in-situ measure ments and a record of the separation in the form of a chromatogram. While all modern laboratories are capable of drug analysis by conventional TLC, only those laboratories equipped with the necessary instrumentation for HPTLC have this option. Preparative TLC is used when one or more components of a sample are required in bulk for further studies, such as for identification by NMR, MS, FTIR, etc. Layers on preparative TLC plates are thicker than on conventional plates to allow higher sample loading. Particle size may also be larger. In the basic TLC experiment, the sample is applied to the layer (stationary phase) as a spot or band near to the bottom edge of the layer. The separation is carried out in a closed chamber either by either contacting the bottom edge of the layer with the mobile phase, which advances through the layer by capillary forces as in Fig. 1, or by forcing the mobile phase to move through the layer at a controlled velocity by means of an external pressure source or centrifugal force. A separation of the sample results from the different rates of migration of the sample components in the direction travelled by the mobile phase, dependent upon their distribution between the stationary and mobile phases. After development and evaporation of the mobile phase, the sample compo nents are separated in space, their position and quantity being determined by visual evaluation or in-situ scanning densitometry aided by the formation of easily detected derivatives by post-chromatographic chemical reactions, as required. The distance the sample has moved along the plate is then noted and is compared with the distance moved by the mobile phase. This is referred to as the retardation factor, or RF value, and is the fundamental parameter used to characterise the position of a sample zone in a TLC chromatogram. For linear development it represents the ratio of the distance migrated by the sample compared to the distance travelled by the solvent front:
where ZX is the distance travelled by the sample from its origin, (Zf - Z0) is the distance travelled by the mobile phase from the sample origin, Zf is the distance travelled by the mobile phase measured from the mobile phase level at the start of the separation, and Z0 is the distance from the sample origin to the mobile phase level at the start of the separation. The boundary conditions for RF values are 1>RF>0. The RF value is generally calculated to two decimal places. Some authors prefer to tabulate values as whole numbers, as hRF values equivalent to 100RF, in which case values are between 0 and 100.
Standards of substances anticipated to be present are normally analysed alongside samples in order to compare their spatial location relative to samples and their response to the detection method used. RF values for standards are compared with those for samples. Separations by column liquid chromatography (HPLC) and TLC occur by essentially the same physical process. The two techniques are frequently considered as competitors, when it would be more realistic to consider them as complementary. TLC methods are most effective for the low cost analysis of a large number of samples (e.g. drug screening in biological fluids and tissues, determination of the botanical origin and potency of traditional herbal medicines, stability testing and content uniformity testing), for the rapid analysis of samples that require minimum sample clean up, or where TLC allows a reduction in the number of sample preparation steps (e.g. analysis of samples containing compo nents that remain sorbed to the stationary phase or contain suspended microparticles). TLC also provides advantages for the analysis of substances with poor detection characteristics that require post-chromatographic chemical treatment for detection. In other cases, HPLC methods are generally preferred, particularly if the sample matrix is very complex and/or sample components have very similar distribution coefficients, for separations by size exclusion and ion-exchange chromatography, and for trace analysis using selective detectors unavailable for TLC.
Figure 1 Illustration of a TLC separation. The sample mixture is spotted at one end of the support plate and placed in the mobile phase. The mobile phase moves across/through the stationary phase carrying the components of the sample with it. The components of the sample partition between the mobile phase and stationary phase. Assuming the correct conditions are chosen, this results in a separation of substances on the plate.
Figure 2 Time line depicting the evolution of modern TLC.
