Introduction and History

Introduction to Thin-layer Chromatography

The basic TLC procedure has largely remained unchanged over the last fifty years. It involves the use of a thin, even sorbent layer, usually about 0.10 to 0.25 mm thick, applied to a firm backing of glass, aluminium or plastic sheet to act as a support. Of the three, glass has always proved the most popular, although aluminium and plastic offer the advantage that they are flexible and can more easily be cut to any size with minimal disruption to the sorbent layer. Numerous sorbents have been used, some more successfully than others, including silica gel, cellulose, aluminium oxide, polyamide and chemically bonded silica gels. The sample is dissolved in an appropriate solvent and applied as spots or bands along one side of the sorbent layer approximately 1 cm from the edge. An eluent (single solvent or solvent mixture) is allowed to flow by capillary action through the sorbent starting at a point just below the applied samples. Most commonly this is achieved by using a glass rectangular tank in which the eluent is poured to give a depth of about 5 mm. The plate is placed in the tank or chromatography chamber and the whole covered with a lid. As the eluent front migrates through the sorbent, the components of the sample also migrate, but at different rates, resulting in separation. When the solvent front has reached a point near the top of the sorbent layer, the plate or sheet is removed and dried. The spots or bands on the developed layer are visualised, if required, under UV light or by chemical treatment or derivatisation. For quantitative determinations, zones can be removed or eluted from the layer, or the plate can be scanned at pre-determined wavelengths without disturbing the layer surface. The modern use of TLC has seen a strong move in the direction of plate scanning and video imaging as a means of providing sensitive and reliably accurate results and a more permanent record of the chromatogram. This is in addition to its obvious labour saving aspect and chemically "clean" approach.

Although TLC is an analytical method in its own right, it is also complimentary to other chromatographic techniques and spectroscopic procedures. Results obtained with TLC can often be transferred to HPLC or vice versa with some adjustment in eluting solvent conditions. For multi-component samples (e.g. pesticides in water), fractions of interest from an HPLC separation can be collected and subsequent re-chromatography of these on HPTLC can give a "fine tuned" separation of the components of the fractions. Thin-layer chromatography has been successfully hyphenated with high performance liquid chromatography (HPLC), mass spectroscopy (MS), Fourier transform infra-red (FTIR), and Raman spectroscopy, to give far more detailed analytical data on separated compounds. Even the UV/visible diode array technique has been utilised in TLC to determine peak purity or the presence of unresolved analytes.

Undoubtedly TLC is a modern analytical separation method with extensive versatility, much already utilised, but still with great potential for future development into areas where research apparently is only just beginning.

2 History of TLC

Although column chromatography can be traced to its discoverer, the Russian botanist, Tswett in l903, it was not until l938 that separations on thin-layers were achieved when Izmailov and Shraiber, looking for a simpler technique, which required less sample and sorbent, separated plant extracts using aluminium oxide spread on a glass plate. The sorbent was applied to a microscope slide as a slurry, giving a layer about 2 mm thick. The sample (plant extracts) was applied as droplets to the layer. The solvent (methanol) was then added dropwise from above on to the applied spots and a series of circular rings were obtained of differing colours on the layer. Circular TLC was born, and Izmailov and Shraiber named this new technique "drop chromatography".

In l949 Meinhard and Hall used a starch binder to give some firmness to the layer, in order to separate inorganic ions, which they described as "surface chromatography". Further advances were made in l95l by Kirchner et al., who used the now conventional ascending method, with a sorbent composed of silicic acid, for the separation of terpene derivatives, describing the plates used as "chromatostrips". In l954, Reitsema used much broader plates and was able to separate several mixtures in one run. Surprisingly it was some time before the advantages of this development were recognised.

However, from l956 a series of papers from Stahl appeared in the literature introducing "thin-layer chromatography" as an analytical procedure, describing the equipment and characterisation of sorbents for plate preparation. Silica gel "nach Stahl" or "according to Stahl" became well known, with plaster of Paris (calcium sulphate) being used as a binder and TLC began to be widely used. In l962, Kurt Randerath's book on TLC was published, followed by those of Stahl and co-workers, entitled 'Thin-Layer Chromatography – A Laboratory Handbook' (1965), and Kirchner's, 'Thin-Layer Chromatography' (1967). Then, in l969 a 2nd edition of Stahl's book appeared which was greatly expanded. These authors showed the wide versatility of TLC and its applicability to a large spectrum of separation problems and also illustrated how quickly the technique had gained acceptance throughout the world. (By 1965 Stahl could quote over 4500 publications.) With Stahl's publication the importance of factors such as controlling the layer thickness, layer uniformity, the binder level and the standardisation of the sorbents as regards pore size and volume, the specific surface area and particle size, were recognised as crucial to obtaining highly reproducible, quality separations.

Commercialisation of the technique began in 1965 with the first pre-coated TLC plates and sheets being offered for sale. TLC quickly became very popular with about 400–500 publications per year appearing in the late 1960s as it became recognised as a quick, relatively inexpensive procedure for the separation of a wide range of sample mixtures. As the range and reliability of commercial plates/sheets improved, standard methods for analysis appeared throughout industry. It soon became evident that the most useful of the sorbents was silica gel, particularly with an average pore size of 60 Å, and it was on this material that the commercial companies centred their attention. Modifications to the silica gel began with silanisation to produce reversed-phase layers. This opened up a far larger range of separation possibilities based on a partition mechanism, compared with adsorption as used in most previous methods.

Up to this time quantitative TLC was fraught with experimental error. However, the introduction of commercial spectrodensitometric scanners enabled the quantification of analytes...