CHAPTER 1
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 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 first to recognise the advantage of using a smaller average particle size of silica gel (about 5–6 µm) in the preparation of TLC plates. He compared the effect of particle size on development time, Rf values and plate height. 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. The technique could now be made fully instrumental to give accuracy comparable with HPLC. Commercially the plates were first called "nano-TLC" plates by the manufacturer, (Merck), but this was soon changed to the designation "HPTLC". In 1977 the first major HPTLC publication appeared, simply called "HPTLC high performance thin-layer chromatography" edited by Zlatkis and Kaiser. In this volume Halpaap and Ripphahn described their comparative results with the new 5 x 5 cm HPTLC plates versus conventional TLC for a series of lipophilic dyes. Bonded phases then followed in quick succession.
Reversed-phase HPTLC was reported in 1980 by Halpaap et al. and this soon became commercially available as pre-coated plates. In 1982 Jost and Hauck reported an amino (NH2-) modified HPTLC plate, which was soon followed by cyano-bonded (1985) and diol-bonded (1987) 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. 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 field 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 ligand exchange principle and is produced commercially either as a TLC or HPTLC plate. Günther has reported results with amino-acids and derivatives on the TLC plate and Mack and Hauck similarly with their HPTLC equivalent.
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 4–5 µm 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.
CHAPTER 2
Sorbents and TLC Layers
1 Sorbent Selection
Introduction
There are at least 25 inert materials that are available as sorbents in TLC, some of which have been more widely used than others. A number of the more important ones will be reviewed in this chapter. Clearly for optimum separations, it is important that the correct material is chosen. Some sorbents have a specific range of application (e.g. silica gel impregnated with caffeine for polyaromatic hydrocarbons, or silica gel impregnated with a chiral selector for the separation of enantiomers of amino-acids and derivatives). By contrast silica gel or aluminium oxide are used for a wide range of applications. Silica gels and aluminas can also be split into a number of distinct, separate sorbents depending on pore size, particle size, and pH. Before choosing the sorbent, consideration must be given to the compounds to be separated. Characteristics, such as the polarity, solubility, ionisability, molecular weight, shape and size of the analytes are all important in deciding on a separation mechanism, and hence largely define both the type of sorbent and the solvents used both for the preparation of the sample and in development.
In 1973 Scott examined over 1100 papers to determine which sorbents were the most regularly used in TLC. Silica gel was by far the most popular (~64%), followed by cellulose (~9%), and alumina (~3%). Since then silica gel has remained the most widely used, but noticeable changes have occurred with the appearance of chemically bonded phases which have opened up a new range of separation possibilities. The newer stationary phases have tended for the most part to address specific areas of separation where either the resolution of sample components was poor or non-existent. As the lists of applications for some sorbents is extensive, it is better to refer to the excellent bibliographies or abstract services that are available for TLC (e.g. Camag Bibliography Service) when a specific method from the literature is required. If this is not available to the user or a new or improved procedure is needed, then the basic information in Table 1 will be of help to ensure that the optimum sorbent for the type of separation is chosen.
1.2 Silica Based Sorbents
1.2.1 Silica Gel
Silica gel, also called silicic acid and kieselgel, is a white amorphous porous material, usually made by precipitation from silicate solutions by addition of acid. The process is by no means simple as polysilicic acids are formed by polycondensation. So-called "primary particles" appear. As the particles grow, water is eliminated and gel formation takes place. The control of the temperature and pH during this stage will have a marked bearing on the quality of the gel formed. Colloidal particles thus develop, which further condense and shrink to form a three dimensional network described as a hydrogel (see Figure 1). After washing and heating (~120 °C) an amorphous hard but porous gel is formed, called a xerogel or silica gel. It is this xerogel that is used for TLC.
The structure is held together by bonded silicon and oxygen, termed siloxane groups. Residual hydroxyl groups on the surface account for much of the adsorptive properties of silica gel giving it unique separation characteristics. These "active sites" can vary according to their local environment. Three types of hydroxyl group are possible as shown in Figure 2. The most prolific is the single hydroxyl group bonded to a silicon atom, which is linked to the silica gel matrix via three siloxane bonds. The second type is where two hydroxyl groups are bonded to a single silicon atom, often called a geminal hydroxyl group. The third type, which is much more rare, is a bonding of three hydroxyl groups to one silicon atom. Only a single siloxane bond binds this group to the silica gel matrix.
However, the available "active" surface is somewhat more complicated by the presence of water that hydrogen bonds to the surface hydroxyl groups. Figure 3 shows that there are quite a number of different ways hydrogen-bonding can occur with water. Even multi-layers of water, physically adsorbed, are possible.
The hydration of the gel for TLC is considered to be 11–12% water when the relative humidity is 50% at 20 °C. Such a gel is normally ready for use requiring no pre-activation. Activation is only necessary if the TLC plate has been exposed to high humidity, and then only requires heating up to 105 °C for 30 minutes followed by cooling in a clean atmosphere at 40–50% relative humidity. The water is held in the structure either as physically adsorbed or hydrogen-bonded water, the latter being more firmly held. As proof of this, desorption of hydrogen-bonded water requires 10 kcal mol-1 whereas physically bound water requires, 6.6–8.2 kcal mol-1 activation energy.
The synthetic nature of silica gel for chromatography enables the careful control of pore size, pore volume and particle size. Pore size varies from 40 to 150 Å for commercial pre-coated TLC plates with one notable exception of 50 000 Å for special applications. The range of particle size of silica gel for TLC is typically 5 to 40 µm with the average being 10 to 15 µm depending on the manufacturer. This has a large effect on the resolution of sample components. Thus in TLC, as in HPLC, reducing the particle size lowers the height equivalent to a theoretical plate of a peak and hence increases the efficiency. As illustrated in Figure 4, when smaller silica gel particles of 5 to 6 micro]m are used to prepare HPTLC plates, improved resolution results.
Pore size affects selectivity and hence can be used to good effect in altering the migration rates and resolution of sample components. The most common pore sizes used in TLC are 40, 60, 80, 100 Å, with silica gel 60 Å being by far the most popular and versatile of the group. Silica gel 60 Å (commonly called silica gel 60) has been recommended for a wide range of separations throughout industry and research institutions. As water content plays such an important role in the retention of analytes on the chromatographic layer, it is vital that the moisture adsorbed by the silica gel is maintained at a constant level. In Figure 5 water adsorption curves are shown for the range of silica gel pore sizes; 40, 60 and 100 Å. At normal levels of humidity in most laboratories (40–60% relative humidity), the variation in uptake of moisture by silica gel 60 Å has little effect on the migration rates of most sample components. The change in water adsorption over small changes in relative humidity (RH) for silica gel 40 Å is quite marked (from 207–40% over an RH range of 40–60%). This will affect on the migration rates of the sample components, and although with careful control humidity differences can be utilised to improve separations, they can also be a source of problems with respect to reproducibility. Although humidity control is not of so much concern for silica gel 100 Å, the sorbent is consequently less polar chromatographically due to comparatively less moisture adsorption resulting in low migration rates of sample components.
