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CHAPTER 1

Introduction and History
1 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 l 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.1–3 Thin-layer chromatography has
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in l969 a 2nd edition of Stahl’s book appeared which was greatly expanded. giving a layer about 2 mm thick. In l954. much already utilised.14 followed by those of Stahl and co-workers. Kurt Randerath’s book on TLC was published. ‘Thin-Layer Chromatography’ (1967). Tswett in l903. The sample (plant extracts) was applied as droplets to the layer. entitled ‘Thin-Layer Chromatography – A Laboratory Handbook’ (1965). and Izmailov and Shraiber named this new technique ‘‘drop chromatography’’.5 it was not until l938 that separations on thin-layers were achieved when Izmailov and Shraiber.4 Undoubtedly TLC is a modern analytical separation method with extensive versatility. and Raman spectroscopy.8 who used the now conventional ascending method. Circular TLC was born.2 Chapter 1 been successfully hyphenated with high performance liquid chromatography (HPLC). in order to separate inorganic ions. the specific surface area and particle size. 2 History of TLC Although column chromatography can be traced to its discoverer.)18 With Stahl’s publication the importance of factors such as controlling the layer thickness. with plaster of Paris (calcium sulphate) being used as a binder and TLC began to be widely used. describing the plates used as ‘‘chromatostrips’’. quality separations. which required less sample and sorbent.. Even the UV/visible diode array technique has been utilised in TLC to determine peak purity or the presence of unresolved analytes. In l962. from l956 a series of papers from Stahl10–13 appeared in the literature introducing ‘‘thin-layer chromatography’’ as an analytical procedure. which they described as ‘‘surface chromatography’’. with a sorbent composed of silicic acid. the Russian botanist. Surprisingly it was some time before the advantages of this development were recognised.15 and Kirchner’s. mass spectroscopy (MS). Silica gel ‘‘nach Stahl’’ or ‘‘according to Stahl’’ became well known. In l949 Meinhard and Hall used a starch binder to give some firmness to the layer. .7 Further advances were made in l95l by Kirchner et al.16 Then. separated plant extracts using aluminium oxide spread on a glass plate.6 looking for a simpler technique. 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. The sorbent was applied to a microscope slide as a slurry. describing the equipment and characterisation of sorbents for plate preparation. (By 1965 Stahl could quote over 4500 publications. the binder level and the standardisation of the sorbents as regards pore size and volume. Reitsema9 used much broader plates and was able to separate several mixtures in one run.17 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. were recognised as crucial to obtaining highly reproducible. layer uniformity. However. for the separation of terpene derivatives. to give far more detailed analytical data on separated compounds. Fourier transform infra-red (FTIR). but still with great potential for future development into areas where research apparently is only just beginning.

simply called ‘‘HPTLC high performance thin-layer chromatography’’ edited by Zlatkis and Kaiser. . In recent years TLC/HPTLC research has entered the chiral separation field using a number of chiral selectors and chiral stationary phases. Automated multiple development (AMD) made its appearance in 1984 due to the pioneering work of Burger.21 In this volume Halpaap and Ripphahn described their comparative results with the new 5 · 5 cm HPTLC plates versus conventional TLC for a series of lipophilic dyes. particularly with ˚ an average pore size of 60 A. but this was soon changed to the designation ‘‘HPTLC’’. Germany.Introduction and History 3 Commercialisation of the technique began in 1965 with the first pre-coated TLC plates and sheets being offered for sale.20 The technique could now be made fully instrumental to give accuracy comparable with HPLC. standard methods for analysis appeared throughout industry. Modifications to the silica gel began with silanisation to produce reversed-phase layers. The 1980s also saw improvements in spectrodensitometric scanners with full computer control becoming possible. and the development distances on the layers could be reduced. In 1982 Jost and Hauck24 reported an amino (NH2–) modified HPTLC plate. and it was on this material that the commercial companies centred their attention. As the range and reliability of commercial plates/sheets improved. In 1977 the first major HPTLC publication appeared.27 This improvement enabled a marked increase in number and resolution of the separated components. but later integrators achieved this automatically.22 Bonded phases then followed in quick succession. Up to this time quantitative TLC was fraught with experimental error. He compared the effect of particle size on development time.19 By the mid 1970s it was recognised that HPTLC added a new dimension to TLC as it was demonstrated that precision could be improved ten-fold. the introduction of commercial spectrodensitometric scanners enabled the quantification of analytes directly on the TLC layer. compared with adsorption as used in most previous methods. Rf values and plate height. Darmstadt. (Mercka). Initially peak areas were measured manually. Reversed-phase HPTLC was reported in 1980 by Halpaap et al. TLC quickly became very popular with about 400–500 publications per year appearing in the late 1960s as it became recognised as a quick. which is based on a a Merck KGaA.23 and this soon became commercially available as pre-coated plates. including options for peak purity and the measurement of full UV/visible spectra for all separated components. which was soon followed by cyano-bonded (1985)25 and diol-bonded (1987)26 phases. Only one type of chiral pre-coated plate is presently commercially available. In l973 Halpaap was one of the first to recognise the advantage of using a smaller average particle size of silica gel (about 5–6 mm) in the preparation of TLC plates. The next major advance was the advent of HPTLC (High performance thin-layer chromatography). analysis time could be reduced by a similar factor. Commercially the plates were first called ‘‘nano-TLC’’ plates by the manufacturer. However. less mobile phase was required. relatively inexpensive procedure for the separation of a wide range of sample mixtures. This opened up a far larger range of separation possibilities based on a partition mechanism. It soon became evident that the most useful of the sorbents was silica gel.

Liq. 82. Chem. N. E. 1963. 2nd edn. 1. SpringerVerlag. 26. 1988. 1954. Pharmaz. 420. New York. Rdsch. 251–262. Miller and G. Sherma and B.29 At the present time all steps of the TLC process can be computer controlled. . Chromatogr. Reitsema. 2. 2nd edn. Burger. Sci. Netherlands. M. and R. E. J.G. 1958. Anal. 19... UK. Germany. Chichester.E. Springer-Verlag. Fried (eds).E. Pharmazie. ¨ 4. 77–78. 23. Brighton. R. Biol. Thin-Layer Chromatography – A Laboratory Handbook. Kirchner. Stahl. Anal. which has enabled TLC to be hyphenated effectively with Raman spectroscopy. 6. D. Marcel Dekker Inc. 1949. Background interference has been reduced. Chromatogr.. 11. Issaq.F. R.. Springer-Verlag. Chem.. Warsaw Soc. 1978. 5. Wiley-Interscience.A. Section. Stahl (ed). Thin-Layer Chromatography – A Laboratory Handbook. 11. Berlin. Stahl. 2nd edn. Thin-Layer Chromatography. Germany. J. The use of highly sensitive charge coupled device (CCD) cameras has enabled the chromatographer to electronically store images of chromatograms for future use (identity or stability testing) and for direct entry into reports at a later date. Stahl. 1903. 7. 1977. Stahl. 8. 1996. Shraiber. Hall. Farmatisiya. Nat. 5. 12. Anal. J. Randerath in Thin-Layer Chromatography. 1956.H. Chem. Kaiser. Berlin. London. 11. 15. 33–44. Elsevier. vol. Techniques in Chemistry. Kaiser. D. 1. 1959. Stahl (ed). USA. UK. 1989. 3.S. ‘Instrumental Thin-Layer Chromatography/Planar Chromatography’. J. Izmailov and M.. K. E. Brighton. 78.. 20. Keller. 1989. R. 633.J. Amsterdam. Zlatkis. Berlin. in HPTLC high performance thin-layer chromatography. Janchen in Handbook of Thin Layer Chromatography. Stahl (ed). Chemiker-Ztg. A. Angew.4 Chapter 1 ligand exchange principle and is produced commercially either as a TLC or HPTLC ¨ plate. Kirchner. 2. ‘Instrumental Thin-Layer Chromatography/Planar Chromatography’. UK. 9. Commercially available HPTLC plates coated with specially pure 4–5 mm spherical silica gel have added further capabilities to the technique. 2l. 1973. UK. E. 1965. J.. 18.M. Germany. 16. Halpaap. 1969. Proceedings of the International Symposium. 2nd edn. Zlatkis. Proc. ¨ 3. 960. 1969. 1941–1965. Academic Press. XIV. Janchen and H. H. Chem. Kaiser (eds). 12. and resolution further improved. 1961.E. 323.G. E. Tswett. J. Meinhard and N. E. 185. 6. 1951. 3 References 1. Gunther has reported results with amino-acids and derivatives on the TLC plate28 and Mack and Hauck similarly with their HPTLC equivalent. Proceedings of the International Symposium. 646. E.. 73. l4. 17. 1938. E. no.E. minute no. Stahl. A. 13. in Thin-Layer Chromatography. 10. 144. J. K.E. 14.

Zlatkis and R. H. 3. ‘Instrumental High Performance Thin-Layer Chromatography’. 241–253. Jost. Anal. Thin-Layer Chromatography’. R. Gunther. Halpaap and J. 1984. Interlaken. M. Kaiser (eds). Kaiser (ed). 1980..E. 215–240. ‘Instrumental High Performance Thin-Layer Chromatography’. Hauck and W. Hauck. 1977. R. ‘Instrumental High Performance. Kaiser (eds). 1988.. Ripphahn.. Z. 11–30. H.F. 25.E. 23.J. K. 1. A. J. Amsterdam. J. K. 1985. Hauck and W. 26. J. in HPTLC high performance thin-layer chromatography. Herbert. Elsevier. Italy. E. H. 22. 1977. Burger. K. Halpaap. Planar Chromatogr. HPTLC high performance thin-layer chromatography. A. Hauck and H. 27. Studer (eds). Wurzburg.E.E. H. Proceedings of 2nd International Symposium. 1987.E. Chromatogr. 448. Germany. Switzerland. Proceedings of 4th International Symposium. H.E. Hauck and W. 1982. Proceedings of 3rd International Symposium. H. Traitler and A. Krebs and H. HRC and CC.E. Zlatkis and R. 304–308. 25–37.E. H. Mack. 318. 1988. Jost. Elsevier. 83–91. Chem. Netherlands.E. Kaiser ¨ (ed). Amsterdam. 29.Introduction and History 5 21. ¨ 28. . Selvino/Bergamo. Netherlands. 228. R. Kaiser. Jost. 24. 95–125.