Special Methods in TLC

This book would not be complete without a mention of a number of other variations of TLC. These have the following features in common  

The equipment described in the following Sections is found in only a few laboratories. The interested user is therefore advised to examine its appearance and function personally at the manufacturers stands in one of the large trade exhibitions (e.g. Achema, Analytica, In Com). 

As the author has as yet had no practical experience with this type of equipment. Practical Tips are not included in this Chapter. 

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After some years, two of these development methods are still used, but new techniques of performing them have come along (see Chapter 11 Special Methods in TLC ). Ascending development is referred to in the present book as the classical method, as in the author s opinion it offers a wider spectrum of possibilities than horizontal development. Lively discussions continue in TLC expert circles on the subject of which of these two methods of development is the better . But what does better mean Every user must find this out for himself or herself at his or her workplace, often doing this afresh for each task. 

The AMD method of Burger is also based on vertical TLC. This system for automated multiple development of thin-layer chromatograms is discussed in Chapter 11 Special Methods in TLC . This equipment falls into the top price bracket and is included here only for completeness. 

Paper LLC and adsorption TLC are basic parts of all major complex methods in current use and likely to remain so. Capital cost is small, risk of obsolescence is small, and training investment is minimal. One technician should specialize in it, and all others should be familiar with it. Simple columns mainly for adsorption chromatography are necessary for the Brown method and are potentially or actually useful for many methods. The simpler techniques are easily mastered, but for some, considerable know-how is needed, most of which can be gleaned from Brown s papers and symposia discussions (E2, G8, L8, P3). 

The ionised sulphate group makes carotenoid sulphates strongly polar. This results in significant solubility in water with values up to 0.4 mg/ml. The high polarity and solubility properties determine the choice of isolation procedure. Special methods are employed and detailed isolation procedures available [68]. The isolation methods include column chromatography on Sephadex or silica, TLC and reversed phase HPLC. Sulphur analysis is also desirable. Further characterisation is carried out by spectroscopic methods. 

The use of MS as a detector for TLC involves identification of substances in a mixture, as well their separation. A necessary condition in this approach is the purity of a sample within a spot on the chromatogram. A special problem is the large quantity of support and, sometimes, a binding material and mobile-phase residues. Some other factors that might potentially hinder TLC/MS are water, which is physically bound to the support, as well as, in some cases, the organic modifier of the support. A factor which complicates the detection process in TLC/MS may also be the presence of derivatives in a sample peak displayed on a chromatogram. That is why the analyzed sample should be as free of contamination with other additives as possible and the support matrix should be carefully removed before MS analysis. These indications refer, to a lesser degree, to other methods of detection that are performed after total extraction of sample molecules from a sample matrix. It is a widely known fact that most of the... 

Tschesche and co-workers recommend the wedged-tip technique (p. 89) for special separation problems. Should this prove inadequate, descending chromatography can be tried. G5ldel and co-workers [76] have applied this method in cases where hi /-values were too similar Khorlin and co-workers [127,128] and Coleman and Parke [33] have preferred two-dimensional TLC on alumina layers. The last named developed with acetone-chloroform (50 + 50) in the first direction and methanol-33% ammonium hydroxide (80 + 20) in the second. 

An important specialized use of TLC involves the separation, identification, and measurement of radioisotopes. The principal methods used are autoradiography, liquid scintillation counting, and direct scanning using a radiation detector interfaced with a computer for data collection. Radiolabeled compounds are used in radiotracer methods to follow the course of chemical and biochemical reactions, e.g., the study of pesticide metabolism in plants and animals (205), and drugs in humans. 

In methods like TLC, validation must be performed in more levels. In the basic level, a scanner must be checked and calibrated according to the manufacturer s specifications. In the second level, it must be validated with a special test plate. This must be done at least once in a year, in order to obtain data about mechanical robustness, repeatability of measurements, monochromator accuracy, baseline noise, and the signal-to-noise ratio. This test plate is prepared by a vendor, and its purpose is detection of any possible malfunction. Too often, use of this informative but time-consuming and complicated test operation is questionable, especially because people in the laboratory are usually not able to repair a scanner, so a service-call is required. In order to know the actual quality of a scanner, a user must prepare a simpler procedure. This procedure can use a part of a vendor test plate, but it is better for each user to prepare an additional in-house test plate. The purpose of such a test is to check the quality of a scanner under working conditions on a daily basis. For accurate results, a third level may be introduced. The best way is to make a system-suitability test on each plate, selecting one spot on a plate, and measuring it with the working paramaters. 

Many natural products are charged substances, and can be isolated by IEC methods. Dufresne has published a comprehensive review describing various resins and column operating conditions applicable to purification of natural products from fermentation broths or crude extracts.168 Among natural products, antibiotics are of special interest due to their widespread use in humans and animals. Sample cleanup by IEC prior to analysis by other LC methods for quantitative determination of antibiotics in biological fluids is frequent.I69171 Also, IEC followed by TLC appears useful for the quantitation of fumonisin Bl, a mycotoxin found in agricultural products.172... 

The adsorbent slurry may be applied to the TLC plates by means of several methods. For analytical work, the plates are best prepared with a special TLC adsorbent applicator which provides uniform layers and can be often adjusted to various thicknesses from 250 pm (normal) up to 500 or 1000 pm for preparative separations. The preparation of plates with a typical applicator is shown in Fig.3.1. These applicators are available from a number of suppliers of TLC equipment including most of those mentioned in Table 3.2. After the slurry has been applied, the plates are dried in air overnight or in a warm oven at 80-90°C for ca. 30 min. The dry plates are stored in a dust-free cupboard for further use. Portable cabinets for plate storage are available from a number of suppliers. 

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