Principle of TLC

Thin layer chromatography (TLC), also known as planar chromatography, is an invaluable method used in chemistry and biochemistry, complementary to HPLC while having its own specificity. Although these two methods are applied differently, the principle of separation and the nature of the phases remain the same. Cheap and sensitive, this technique that is simple to use, can be automated. It has become essential principally since it is possible to undertake several separations in parallel. The development of automatic applicators and densitometers have led to nano-TLC, also called HPTLC, a highly sensitive technique which can be hyphenated with mass spectrometry. 

There are three steps to conduct a separation with this technique  

A small volume of sample (between a few nanolitres to a few microlitres), dissolved in a volatile solvent, is deposited close to the bottom of the plate as a small 

Chemical Analysis Second Edition Francis and Annick Rouessac 

The mobile phase rises up the stationary phase by capillarity, moving the components of the sample at various rates because of their different degrees of interaction with the matrix and solubility in the solvent. Their separation may be complete in a few minutes. When the solvent front has travelled a sufficient distance (several centimetres), the plate is withdrawn from the chamber, the position attained by the mobile phase is immediately noted then it is evaporated. 

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Thin layer chromatography (TLC) and paper chromatography (PC), which together comprise flat-bed or planar chromatography, are the easiest of all chromatographic methods to perform, and they require only simple and inexpensive equipment. Many of the techniques and principles of TLC and PC are the same. They both readily provide qualitative analytical information, and, with optimization of techniques and materials, can give quantitative data as well. 

An apparent advantage of HPLC over TLC is its easy access to automation. Several approaches to automate the TLC process have been discussed (8,9). However, changing the off-line principle of TLC to an on-line closed system, would combine the limitations of TLC with those of HPLC. Automation of the individual steps of the TLC process, as set forth in this chapter, appears to be the better choice. Besides, one should not overlook, that with a stepwise automated TLC workstation, more samples can be processed in 8 hours than with a fully automated HPLC system within 24 hours. 

In the course of mixture separation, the composition and properties of both mobile phase (MP) and stationary phase (SP) are purposefully altered by means of introduction of some active components into the MP, which are absorbed by it and then sorbed by the SP (e.g. on a silica gel layer). This procedure enables a new principle of control over chromatographic process to be implemented, which enhances the selectivity of separation. As a possible way of controlling the chromatographic system s properties in TLC, the pH of the mobile phase and sorbent surface may be changed by means of partial air replacement by ammonia (a basic gaseous component) or carbon dioxide (an acidic one). 

The two examples that have been given are simple and basic, and illustrate the principles of a TLC separation. Ion exchange material can also be bonded to the silica, allowing ionic interactions to be dominant in the stationary phase and, thus. 

The power of TLC is in its flexibility as a problem solving tool. As the problems in analysis become more complicated the sophistication by which we approach those problems is ever increasing. However, it behooves us as analytical chemists not to forget our fundamental training in chemistry and to apply those principles to today s problems. It is just this feature that the reader will find instilled into this book. 

The principle of 2-D TLC separation is illustrated schematically in Figure 8.4. The multiplicative law for 2-D peak capacity emphasizes the tremendous increase in resolving power which can be achieved in theory, this method has a separating capacity of n, where n is the one-dimensional peak capacity (9). If this peak capacity is to be achieved, the selectivity of the mobile phases used in the two different directions must be complementary. 

On the basis of the principle of grafted TLC, reversed-phase (RP) and normal-phase (NP) stationary phases can also be coupled. The sample to be separated must be applied to the first (2.5 cm X 20 cm) reversed-phase plate (Figure 8.16(a)). After development with the appropriate (5ti 5yi) mobile phase (Figure 8.16(b)), the first plate must be dried. The second (20 cm X 20 cm) (silica gel) plate (Figure 8.16(c)) must be clamped to the first (reversed-phase) plate in such a way that by use of a strong solvent system (Sj/, SyJ the separated compounds can be transferred to the second plate (Figure 8.16(d)). Figure 8.16(e) illustrates the applied, re-concentrated... 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

Principles and Characteristics Many of the planar chromatography methods rely on fluorescence detection to achieve the required identification limits exploitation of sensitive and selective derivatisation reactions is of considerable importance. Most TLC scanning densitometers can be operated in the fluorescence mode and are able to record in situ excitation spectra of TLC... 

Principles and Characteristics Both mid-IR (2.5-50 p.m) and near-IR (0.8-2.5 p.m) may be used in combination to TLC, but both with lower sensitivity than UV/VIS measurements. The infrared region of the spectrum was largely ignored when the only spectrometers available were the dispersive types. Fourier-transform instruments have changed all that. Combination of TLC and FTIR is commonly approached in two modes ... 

Principles and Characteristics The prospects of Raman analysis for structural information depend upon many factors, including sample scattering strength, concentration, stability, fluorescence and background scattering/fluorescence from the TLC substrate. Conventional dispersive Raman spectroscopy has been considered as a tool for in situ analysis of TLC spots, since most adsorbents give weak Raman spectra and minimal interference with the spectra of the adsorbed species. Usually both silica and cellulose plates yield good-quality conventional Raman spectra, as opposed to polyamide plates. Detection limits for TLC fractions... 

High-performance thin-layer chromatography (HPTLC) has become widely used and while it follows the same principles as TLC, it makes use of modern technology including automatic application devices and smaller plates, which allow for better sensitivity. 

As will be shown later, the former three mechanisms mentioned above are applicable to TLC separation of polymers. From the standpoint of TLC applied to polymer separation, the partition mechanism may be better expressed by phase-separation or precipitation mechanism, as will be explained in Section II.3. It should be noted that all these mechanisms are generally present during a chromatographic separation. Therefore, one mechanism should be made to be predominant for a given separation aim. This can, in principle, be done by properly selecting the developer and adsorbent. However, such a selection is the major problem in application of TLC, especially, to polymer separation, and the following three sections will be devoted to describing the rules that have been established to solve this problem. 

The latter three are obtained by solution polymerization technique with alkyllithium initiator through the anionic mechanism. For these materials, the analysis of block sequences is also an interesting subject in the area of TLC application. However, because a somewhat different principle has to be applied to achieve separation by the difference in block sequences, this subject will be discussed in a subsequent section (cf. Section IV.2.). 

For the quantitation of chromatograms, the development condition found by the conventional TLC was transferred to another TLC system which uses a thin quartz rod coated with silica gel of 75 p thickness and may be called thin-layer-FID chromatography . The TLC system is equipped with a flame ionization detector (FID) and commercially available as a complete set (Iatron thinchrograph model TFG-10, Iatron Co., Ltd., Tokyo). The principle of sample scanning and device for FID are almost the same as those worked out by Padley57), Szakasits et al, and... 

Figure 5.3—Principle of two-dimensional TLC. The use of distinctly different mobile phases is recommended for a better separation, a) Deposition of three standards and an unknown b) Migration with the first solvent c) drying and rotation of the TLC plate d) migration with the second solvent. Conclusion . v is probably a, but the impurity is not b or c.

The principle of gas chromatography (GC) is similar to that of liquid chromatography or TLC, in that compounds in mixtures are separated from each other based on their affinity for a resin. GC is performed on volatile... 

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