CHAPTER 1

Introduction and History

1 Introduction to Thin-layer Chromatography
The basic TLC procedure has largely remained unchanged over the last fty years. It involves the use of a thin, even sorbent layer, usually about 0.10 to 0.25 mm thick, applied to a rm 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 exible 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 ow 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 ne tuned separation of the components of the fractions.13 Thin-layer chromatography has
1

Chapter 1

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.4 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 l903,5 it was not until l938 that separations on thin-layers were achieved when Izmailov and Shraiber,6 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 rmness to the layer, in order to separate inorganic ions, which they described as surface chromatography.7 Further advances were made in l95l by Kirchner et al.,8 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, Reitsema9 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 Stahl1013 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 Randeraths book on TLC was published,14 followed by those of Stahl and co-workers, entitled Thin-Layer Chromatography A Laboratory Handbook (1965),15 and Kirchners, Thin-Layer Chromatography (1967).16 Then, in l969 a 2nd edition of Stahls book appeared which was greatly expanded.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. (By 1965 Stahl could quote over 4500 publications.)18 With Stahls 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 specic surface area and particle size, were recognised as crucial to obtaining highly reproducible, quality separations.

Introduction and History

Commercialisation of the technique began in 1965 with the rst pre-coated TLC plates and sheets being offered for sale. TLC quickly became very popular with about 400500 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 A, and it was on this material that the commercial companies centred their attention. Modications 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 quantication of analytes directly on the TLC layer. Initially peak areas were measured manually, but later integrators achieved this automatically. The next major advance was the advent of HPTLC (High performance thin-layer chromatography). In l973 Halpaap was one of the rst to recognise the advantage of using a smaller average particle size of silica gel (about 56 mm) in the preparation of TLC plates. He compared the effect of particle size on development time, Rf values and plate height.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, analysis time could be reduced by a similar factor, less mobile phase was required, and the development distances on the layers could be reduced.20 The technique could now be made fully instrumental to give accuracy comparable with HPLC. Commercially the plates were rst called nano-TLC plates by the manufacturer, (Mercka), but this was soon changed to the designation HPTLC. In 1977 the rst major HPTLC publication appeared, simply called HPTLC high performance thin-layer chromatography edited by Zlatkis and Kaiser.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.22 Bonded phases then followed in quick succession. Reversed-phase HPTLC was reported in 1980 by Halpaap et al.23 and this soon became commercially available as pre-coated plates. In 1982 Jost and Hauck24 reported an amino (NH2) modied HPTLC plate, which was soon followed by cyano-bonded (1985)25 and diol-bonded (1987)26 phases. The 1980s also saw improvements in spectrodensitometric scanners with full computer control becoming possible, including options for peak purity and the measurement of full UV/visible spectra for all separated components. Automated multiple development (AMD) made its appearance in 1984 due to the pioneering work of Burger.27 This improvement enabled a marked increase in number and resolution of the separated components. In recent years TLC/HPTLC research has entered the chiral separation eld using a number of chiral selectors and chiral stationary phases. Only one type of chiral pre-coated plate is presently commercially available, which is based on a
a

Merck KGaA, Darmstadt, Germany.

Chapter 1

ligand exchange principle and is produced commercially either as a TLC or HPTLC plate. Gunther has reported results with amino-acids and derivatives on the TLC plate28 and Mack and Hauck similarly with their HPTLC equivalent.29 At the present time all steps of the TLC process can be computer controlled. 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. Commercially available HPTLC plates coated with specially pure 45 mm spherical silica gel have added further capabilities to the technique. Background interference has been reduced, and resolution further improved, which has enabled TLC to be hyphenated effectively with Raman spectroscopy.

3 References
1. K. Burger, Instrumental Thin-Layer Chromatography/Planar Chromatography, Proceedings of the International Symposium, Brighton, UK, 1989, 3344. 2. R.E. Kaiser, Instrumental Thin-Layer Chromatography/Planar Chromatography, Proceedings of the International Symposium, Brighton, UK, 1989, 251262. 3. D. Janchen and H.J. Issaq, J. Liq. Chromatogr., 1988, 11, 19411965. 4. D. Janchen in Handbook of Thin Layer Chromatography, 2nd edn, J. Sherma and B. Fried (eds), Marcel Dekker Inc., New York, USA, 1996, 144. 5. M. Tswett, Proc. Warsaw Soc. Nat. Sci., Biol. Section, 1903, l4, minute no. 6. 6. N.A. Izmailov and M.S. Shraiber, Farmatisiya, 1938, no. 3, 1. 7. J.E. Meinhard and N.F. Hall, Anal. Chem., 1949, 2l, 185. 8. J.G. Kirchner, J.M. Miller and G.E. Keller, Anal. Chem., 1951, 23, 420. 9. R.H. Reitsema, Anal. Chem., 1954, 26, 960. 10. E. Stahl, Pharmazie, 1956, 11, 633. 11. E. Stahl, Chemiker-Ztg, 1958, 82, 323. 12. E. Stahl, Pharmaz. Rdsch., 1959, 2, 1. 13. E. Stahl, Angew. Chem., 1961, 73, 646. 14. K. Randerath in Thin-Layer Chromatography, Academic Press, London, UK, 1963. 15. Thin-Layer Chromatography A Laboratory Handbook, E. Stahl (ed), Springer-Verlag, Berlin, Germany, 1965. 16. J.G. Kirchner, Thin-Layer Chromatography, 2nd edn, Techniques in Chemistry, vol. XIV, Wiley-Interscience, Chichester, UK, 1978. 17. Thin-Layer Chromatography A Laboratory Handbook, 2nd edn, E. Stahl (ed), Springer-Verlag, Berlin, Germany, 1969. 18. E. Stahl, in Thin-Layer Chromatography, 2nd edn, E. Stahl (ed), SpringerVerlag, Berlin, Germany, 1969, 5. 19. H. Halpaap, J. Chromatogr., 1973, 78, 7778. 20. A. Zlatkis, R.E. Kaiser, in HPTLC high performance thin-layer chromatography, A. Zlatkis, and R.E. Kaiser (eds), Elsevier, Amsterdam, Netherlands, 1977, 12.

Introduction and History

21. HPTLC high performance thin-layer chromatography, A. Zlatkis and R. E. Kaiser (eds), Elsevier, Amsterdam, Netherlands, 1977. 22. H. Halpaap and J. Ripphahn, in HPTLC high performance thin-layer chromatography, A. Zlatkis and R.E. Kaiser (eds), Elsevier, Amsterdam, Netherlands, 1977, 95125. 23. H. Halpaap, K.F. Krebs and H.E. Hauck, J. HRC and CC, 1980, 3, 215240. 24. H.E. Hauck and W. Jost, Instrumental High Performance, Thin-Layer Chromatography, Proceedings of 2nd International Symposium, R.E. Kaiser (ed), Interlaken, Switzerland, 1982, 2537. 25. H.E. Hauck and W. Jost, Instrumental High Performance Thin-Layer Chromatography, Proceedings of 3rd International Symposium, R.E. Kaiser (ed), Wurzburg, Germany, 1985, 8391. 26. H.E. Hauck and W. Jost, Instrumental High Performance Thin-Layer Chromatography, Proceedings of 4th International Symposium, R.E. Kaiser, H. Traitler and A. Studer (eds), Selvino/Bergamo, Italy, 1987, 241253. 27. K. Burger, Z. Anal. Chem., 1984, 318, 228. 28. K.J. Gunther, J. Chromatogr., 1988, 448, 1130. 29. M. Mack, H.E. Hauck and H. Herbert, J. Planar Chromatogr., 1988, 1, 304308.