2

If you believe that Chapter 1 has been overwhelmingly difficult, you might want to skip this one and continue with Chapter 3, because now we go even deeper into the background of what is going on during the TLC process. But don’t worry; even though we think that a good theoretical foundation will make your work with TLC much easier, you can still follow the next chapters without it. Coming back later to this part of the book or exploring the primary literature on the subject is always an option.

This chapter first offers a closer look at general chromatographic principles. Then, a discussion of special parameters of TLC is provided. Many good books on theoretical aspects of TLC have been written,^1–4and this chapter is not intended to be a substitute. We are limiting mathematical approaches and equations to an absolute minimum. We know that we are not comprehensive. It is our intention to offer some guidance to those who want to really study and understand TLC but don’t know where to begin.

General Chromatographic Principles

In Chapter 1, we introduced a simple model of the chromatographic separation. Now let’s look in more detail at the molecular processes that are the basis of it. First, it is necessary to understand how retention of molecules works. Next, we draw attention to the effects of diffusion. Finally, we want to discuss how a chromatogram is recorded and quantitatively evaluated.

Retention Mechanisms

Chromatography depends on the “affinity” of sample components to the stationary and the mobile phases of the chromatographic system. For each substance in a given chromatographic system, a so-called partition coefficient K (Eq. 2.1) can be defined as the ratio of substance concentration in the stationary phase (c_s) and substance concentration in the mobile phase (c_m)^c.

^aTo be more exact, the chemical activities of the substance in stationary and mobile phase should be used.

Let us assume stationary and “mobile” phases are in a container, such as a separatory funnel. The sample, consisting of one substance only, is introduced. Provided it is soluble in both, the sample would distribute itself between the two phases. After a certain time, an equilibrium described as K will be established. In chromatography, the mobile phase is in motion, but nevertheless the sample attempts to reach partition equilibrium between the two phases (Fig. 2–1).

While in the mobile phase, a molecule moves with the flow. While on/in the stationary phase, a molecule generally remains immobile; it is retained with respect to the flow of the mobile phase. Overall, some substances seem to move faster than others, leaving the column with shorter retention time in HPLC or advancing to higher R_F values on the TLC plate. Actually, all substances move with the same velocity while in the mobile phase. Some are just less likely to be dissolved in it, thus migrating less often per time unit. The question is, why?

In gas chromatography, temperature predominantly controls, whether a sample evaporates into the gas phase or resides in the stationary phase and is retained.

In liquid chromatography, it is more complicated because the sample molecule always interacts with its surroundings. This means it is either dissolved in a liquid or attached to the surface of a solid. There are various types of interactions at the molecular level, which can contribute to retention. It is the combination of solubility in the mobile phase and solubility in or adsorptivity on the stationary phase that determines its degree.

What are the mechanisms most relevant for TLC, keeping in mind that in practice often a mixture of different effects is observed?

Figure 2–1 Schematic retention in liquid chromatography: (A) adsorption, (B) partition. Blue bullets, sample molecules; red arrows, mobile phase; purple surface, silica gel; yellow layer, bonded liquid stationary phase (C18 for example).

Adsorption

In adsorption chromatography, the stationary phase is generally a solid, at the surface of which a substance can engage in a variety of specific and nonspecific interactions. The resulting adsorption lowers the surface energy of the adsorbent. Polar adsorbents such as silica gel, aluminum oxide, or other inorganic materials feature a very large “internal” surface because of their porous structure. This surface is heterogeneous. Active sites are available for specific association with sample molecules based for instance on dipole–dipole or Lewis acid–base interaction, hydrogen bonding, or ionic interaction. The degree of retention is based on the type and number of functional groups of the sample molecule and on its steric structure (Fig. 2–2A). In so-called normal or straight phase chromatography, the mobile phase is “organic” and less polar than the adsorbent. Mobile phase molecules are also adsorbed on the stationary phase and therefore in competition with sample molecules. Mathematical description of adsorption is rather difficult. The two important approaches, the competition model (Snyder⁵ and Soczewinski⁶) and the solvent interaction model (Scott and Kucera7), are reviewed and compared in detail by Poole and Poole² (pp. 375–388). The first model is based on the displacement of adsorbed solvent molecules from adsorption sites on the stationary phase by sample molecules. The second model views the retention mechanism as adsorption process emphasizing sample-mobile phase interactions. Depending on the composition of the mobile phase, both processes take place alternatively.

^bIn some cases, the stationary phase is an immobilized (e.g., chemically bonded) layer of liquid.

Figure 2–2 Retention in TLC: (A) adsorption, (B) partition, (C) complex formation. Red, MP components; blue, sample components; black, stationary phase structure.

In adsorption TLC, the term activity is used to describe a certain property of the adsorbent affecting retention. Activity of the stationary phase is also of fundamental practical importance, because it is strongly influenced by the relative humidity to which the stationary phase was exposed prior to chromatogram development.

Partition

Partition chromatography utilizes the different solubility of a sample component in two immiscible liquid phases. The stationary liquid phase is immobilized on a solid, usually porous support, either by adsorption or by chemical bonds^c.

^cIn countercurrent chromatography, two liquids of different densities are used, of which one 24 is forced through the other by pump action.⁸

Partition systems can be generated dynamically during chromatography if a component of the mobile phase is much stronger adsorbed or wets the surface better than other components. In such a case, we speak of a solvent-generated stationary phase. For example, water is forming a multilayer on cellulose or silica gel when water-saturated mixtures of organic solvents are used as mobile phase. Such systems are suitable for separation of water-soluble polar substances. Acid, base, or a buffer can be added to the mobile phase to control protolysis and to separate the nondissociated form of acidic or basic compounds. Because the surface of the solid support is not directly involved in the separation process, no activity effects are seen (Fig. 2–3). Consequently, reproducibility of the chromatographic results in partition systems is easier to achieve than that obtained with true adsorption systems. On the other hand, it is very difficult to predict relative retention of a given set of compounds (e.g., a mixture of monosaccharides or amino acids). Optimization of the separation tends to be a matter of trial and error.

Modern stationary phases for partition chromatography are chemically bonded onto silica gel. Reversed phases (RP) and polar bonded phases (PBP) can be looked at as a monomolecular liquid film on a solid support. RP systems consist of nonpolar stationary phase and polar aqueous mobile phase. Retention in such systems can be explained with the solvophobic concept of Horvath.⁹ Because of their at least partial hydrophobic nature, organic molecules are “expelled” from the aqueous mobile phase and “incorporated” into the lipophilic stationary phase. On an RP phase (for example, a C8 or a C18), retention depends on aspects of lipophily, such as length of carbon chain, absence of branches, or steric hindrance (Fig. 2–2B). The theoretical description of PBP, such as cyano-, amino-, and diol-bonded phases, is more complicated. Because of their ability to engage in specific interaction with functional groups of the sample, adsorption on the surface of such phases must be considered.

Complex Formation

Ligand exchange is another possible retention mechanism. The basic principle is the reversible formation of coordination compounds between Lewis acids and bases. For separation of unsaturated or polyaromatic compounds, the stationary phase can be impregnated with Lewis acids, such as metal ions (e.g., Ag⁺, Zn²⁺), or strongly electrophilic organic compounds (e.g., picric acid, caffeine) (Fig. 2–2c). Such systems allow separating compounds according to the degree of unsaturation and according to steric effects, like cis/trans configuration or type of substitution. Also, separation of chiral compounds can be accomplished through complex formation. An immobilized chiral selector such as hydroxyproline in the stationary phase can form different diastereomeric coordination compounds with the enantiomers of amino acids. Cu²⁺ ions are used as coordinative centers.

Other Separation Mechanisms

Although not very important for TLC, some other retention mechanisms should be mentioned. Ionic compounds, or those that can be ionized, are separated by ionic interaction, which means the attraction of opposite charges. Retention depends on the number of charges in relation to the size of the ionic species. Ionization is typically controlled by the pH of the mobile phase, and separations are set up for either cations or anions.

Ion-exchange chromatography requires a stationary phase that is able to stoichiometrically replace its counterions with ions of the analyte. Cation exchangers feature functional groups like sulfonate, phosphate, or caboxylate on resins, dextranes, or cellulose support. Anion exchangers carry ammonium or quaternary alkyl ammonium sites. Retention is a function of charge density and polarizability of the involved ions.

Ion-pair chromatography is the more common way to separate electrolytes. The stationary phase is typically a C8 or C18 bonded phase. Adding to the mobile phase ion-pairing reagents, such as sodium alkyl or aryl sulfonates for basic samples or, respectively, tetra alkyl ammonium bromides for acidic samples, results in the formation of ion pairs. They can be separated like, and together with, neutral compounds present in the same sample. Partitioning of the ion pair between stationary and mobile phase is the principal retention mechanism. Another currently preferred model assumes that the ion-pairing reagent becomes part of the stationary phase, either through impregnation prior to chromatography or through absorption out of the mobile phase during chromatography. This way, retention can be seen as dynamic ion exchange.

Size-exclusion chromatography can be used to separate molecules according to their size. The defined pores of the stationary phase allow penetration by molecules up to a specific size (often expressed as molecular weight). The smaller the molecule, the deeper it can diffuse into the pore and the more it is “retained.” Molecules excluded from the pores due to their large size will elute first. Synthetic resins, cross-linked dextranes (Sephadex®), or silica gel can be used as stationary phases.

Inclusion chromatography, as the name says, is based on the formation of inclusions. Certain substances like cyclodextrins form cages of specific dimension and chemical functionality into which the sample molecule can enter. Optical and structural isomers can be separated with this technique.

Efficiency, Diffusion, Selectivity, and Resolution

This section brings us back to the fundamental principle of chromatography, the distribution of the sample between the stationary and the mobile phases. In a very simplistic approach, we can assume that during the time it takes to establish the corresponding equilibrium of concentrations, the mobile phase has advanced a certain distance, called a theoretical plate. This name was taken from distillation where it has a very similar meaning. Physically, the height equivalent to a theoretical plate (HETP, or H) is in the range of 10 to 100μ m. In the rate theory, a different approach to chromatographic separation, H is called efficiency, the ability of a chromatographic system to generate “sharp” peaks.

For separation of two compounds, the chromatographic system must be selective, which means the compounds are retained to a different extent. This is achieved if the partition coefficients of the compounds are different and the selectivity factor α is larger than 1 (Eq. 2.2).

where α is selectivity factor (greater than 1) and K is partition coefficient.

Several theoretical plates must be passed to make a separation quantitative. Unfortunately, whenever the sample molecule is in the mobile phase, it is also subjected to diffusion, which counteracts the efficiency. Let us look at these terms in more detail and discuss how they are calculated and empirically determined.

HETP (or H) and Plate Number (N)

Both terms H (HETP) and N express the efficiency of a chromatographic system. Using Eq. 2.3, one can be converted into the other. L is the length of the column or the developed distance of the TLC plate.

where N is plate number, L is length of system, and H is height equivalent to a theoretical plate.

The plate number can also be directly calculated from the chromatogram assuming a Gaussian peak (Fig. 2–4; Eq. 2.4a and Eq. 2.4b). Either the peak’s base width (W_b) or

Figure 2–4 Characteristics of a Gaussian peak. h, peak height;, standard deviation; W_h, peak width at half height; W_b, base width

Typical efficiencies for various chromatographic systems are compared in Table 2–1. It is desirable to have large plate numbers available to ensure sharp peaks and to be able to separate several compounds with baseline resolution. Large plate numbers can theoretically be achieved with low values for H or long columns or developing distance, but in practice there are physical limits.

Band Broadening

If a sample is injected as a plug (band) into a tube through which a solvent is flowing, diffusion causes the band to broaden significantly. The process, called longitudinal diffusion, is caused by the heat movement of molecules in solution and is particularly important at low flow rates (Fig. 2–5).

In a chromatographic column, additional effects that contribute to band broadening can be observed. (For detailed discussion, see Ref. 3.) When the mobile phase flows through a bed of densely packed small particles, a molecule can take several different paths (Fig. 2–6A). Based on the different length of the individual paths and the different flow rates in paths of different width, molecules advance differently. This process, called eddy diffusion, spreads the initial bandwidth considerably and can only be controlled by the quality of the packing of the chromatographic bed. When looking at a single channel between two particles, we observe that the flow closer to the particle is slower than in the middle of the channel (Fig. 2–6B). This effect, the mobile phase mass transfer, would also be seen in a simple tube as laminar flow (Fig. 2–5). Finally, there are two effects that are actually requirements for chromatography but also contribute to band broadening. Fig. 2–6C illustrates the stagnant mobile phase mass transfer. This effect is used in size-exclusion chromatography. Mobile phase in the pores of a stationary-phase particle is not moving. If a molecule diffuses into the pore, it stops moving relative to a molecule that is still in the flow. The stationary phase mass transfer (Fig. 2–6D) is caused by the possibility that a molecule diffuses into a different depth of the (liquid) stationary phase.

The van Deemter equation (Eq. 2.5a; Fig. 2–7) is of fundamental importance for chromatography because it links the various causes for band broadening with the efficiency H. For detailed discussion, see Refs. 1, 2, and 10.

where H is height equivalent to a theoretical plate, A is constant (eddy diffusion, mobile phase mass transfer), B is constant (longitudinal diffusion), C is constant (stagnant mobile phase, stationary phase mass transfer), and u is flow velocity (cm/s).

In its original form developed to describe GC columns, the van Deemter equation includes three terms. The A term, a constant, primarily depending on the average particle size and homogeneity of the stationary phase, is an expression for the eddy diffusion and mobile phase mass transfer. The B term represents longitudinal diffusion and becomes less important with increasing mobile phase velocity u. The C term describes stagnant mobile phase and stationary phase mass transfer. This term increases with increasing mobile phase velocity. Typically, van Deemter plots have a pronounced minimum for H. The related value for u is called optimum flow rate. In HPLC, the modified Eq. 2.5b is used.

Figure 2–7 Schematic plot of the van Deemter equation.

Figure 2–8 Resolution between two peaks (schematic): t₁, retention time of first peak; t₂, retention time of second peak; W_t1, base width of first peak; W_t2, base width of second peak.

Resolution

The separation of two compounds can be expressed as the resolution (R_s) between the corresponding peaks in the chromatogram (Fig. 2–8; Eq. 2.6)

where R_s is resolution, t_{1 resp 2} is retention time of peaks, and Wt1 resp 2 is base width of peaks.

To separate two components to at least 98%, a resolution of R_s= 1.0 is necessary. Baseline resolution (99.5%) is achieved at R_s= 1.25. A more detailed discussion of this subject is found in Ref. 3. Several factors affect resolution and can therefore be used to control it. Let us look at Eq. 2.7.

where R_s is resolution, αis selectivity factor, N is plate number, and kʹ is capacity factor.

Term a of the equation is the selectivity term and the easiest to control. As we have seen in Eq. 2.1 and Eq. 2.2, it is the proper combination of stationary and mobile phase that is responsible for good resolution in a given experimental setup. The efficiency term b is affected by changing the length of the column (limited by the resulting increase in back pressure), the flow rate of the mobile phase (optimum; see Fig. 2–7), or the column packing (smaller particles). The c term depends on the solvent strength of the mobile phase. “Weaker” solvents yield larger kkʹ values. If kkʹ is initially less than 1, increasing it will improve R_s, but for kkʹ greater than 5, this increase is only marginal because of excessive band broadening.

Quantitative Analysis

So far, we have been concerned about the separation of compounds as a consequence of different retention. As a principal requirement for monitoring separation, the substances of interest must be detectable. The position of the peak maximum in the recorded chromatogram marks the retention time/migration distance of the corresponding substance, and it can be used for identification in qualitative analysis.

After integration of the chromatogram, the height and/or area of the individual peaks can be quantitatively evaluated (Fig. 2–9). The detector output (signal) is either concentration sensitive, responding to a change of mass per unit volume, or mass sensitive, responding to a change of mass per unit time. Peak data of reference substances of known concentration/mass are needed for comparison.

Let us briefly discuss some general characteristics of a detector: The sensitivity of a mass sensitive detector is defined as the quotient of peak area and corresponding sample amount. When no sample is present, the detector puts out a signal, which, plotted against time/position, is called baseline (B). Typically, a baseline is not flat but shows some small fluctuations, called noise (N). Each individual compound has a certain limit of detection (LOD), which is the amount that generates a signal significantly (two to three times) larger than the detector noise. The dynamic range of a detector can be determined by plotting the detector signal against increasing sample amounts. It includes those sample amounts that create a discernible change in the signal at the lower end (noise) and at the upper end (saturation). The peak asymmetry parameter (a/b) indicates tailing (more than 1) or leading (less than 1).

Figure 2–9 Quantitative chromatogram data: M, peak maximum (at t1, retention time; or MD, migration distance); A, peak area; h, peak height; B, baseline; N, noise; a/b, peak asymmetry parameter; LOD, limit of detection.

TLC uses the off-line principle. An analysis can be looked at as a sequence of individual and more or less independent steps. Each of those is characterized by many parameters not available in online separations. In this section, we will focus on chromatogram development. The other steps are discussed from a practical point of view in Chapter 3.

The Flow of the Mobile Phase

The fact that capillary action causes the mobile phase flow in TLC is of fundamental consequence. Unlike GC and HPLC, where the flow within certain limits can easily be adjusted, TLC is “at the mercy” of capillary forces. They follow certain rules but are generally not controllable. We want to understand why things in TLC are and have to be done in certain ways. At the end of this section, we will discuss an option of applying a forced flow. For an in-depth treatment of the subject, please refer to Refs. 1 and 2.

Capillary Flow

The capillary flow can be explained from two starting points leading to a state of lower energy. The stationary phase is an adsorbent, characterized by small particles with high surface energy due to the presence of a huge number of micropores. It also contains cavities or channels between the particles of the layer packing. When the surface of the adsorbent is covered with mobile phase molecules, the energy is lowered. This is one reason for the flow. The other reason originates from the solvent, which attempts to lower its own energy by entering a capillary cavity. Equation 2.8 describes the resulting energy change ΔE as proportional to the surface tension γ and the molar volume V_m of the liquid and inversely proportional to the diameter D of the pores.

where ΔE is energy change, γ is surface tension, V_m is molar volume, and D is pore diameter.

As a consequence, the smaller capillaries of the particles are filled first, and larger pores at and close to the migrating solvent front remain empty for some time. A frontal volume gradient is established, which makes detection of the “true” solvent front difficult.

The movement of the mobile phase front position z_f (see Fig. 2–10) with respect to the immersion line can be described with Eq. 2.9a. The so-called flow constant k takes into account the permeability (K₀) and the average particle size (d_p) of the layer as well as the surface tension (γ), viscosity (η), and contact angle (θ) of the mobile phase.

where z_f is mobile phase front position, k is flow constant, K₀ is permeability, d_p is average particle size,γ is surface tension, η is viscosity, and θ is contact angle.

In Fig. 2–11 experimentally obtained results are plotted for some mobile phases on silica gel. Good agreement with the general equation is seen.

In practice, it is difficult to predict the actual velocity of the mobile phase during the development of the TLC plate because several other effects can change it. If the developing chamber is not saturated, some components of the migrating mobile phase evaporate, slowing down the advancement of the front. In saturated chambers, preadsorption of solvent molecules from the gas phase can speed it up. Viscous solvents such as butanol migrate very slowly.

On reversed phases, the front velocity is generally slower than on normal phases. This is due to the large contact angle (θ) of water on the nonpolar surface of the capillary. When with increasing water content (over 40%) approaches 90 degrees, the flow constant k becomes very small (Eq. 2.9b) and no movement is possible at all. With respect to mobile phase composition, reversed-phase TLC is much less flexible than RP-HPLC.

Figure 2–11 Experimentally obtained plots of the solvent front position on HPTLC Si 60 with increasing development times. Blue diamonds: toluene, ethyl acetate (95:5). Green triangles: TBME, methanol, ammonia (20:2:1). Pink squares: ethyl acetate, methanol, water, formic acid (50:2:3:6).

The principal conclusion from Eq. 2.9a is that the mobile phase in TLC slows down with increasing developing distance until it eventually will come to a halt. Looking back at the van Deemter equation (Eq. 2.5; Fig. 2–7), we can conclude that the consequence will be a dramatic increase of H as soon as the mobile phase velocity drops below u_opt. This effect is particularly important for HPTLC plates, because they feature much denser packing than TLC plates and generate a greater resistance to the flow. How far should we develop a plate for best results? An answer to this question can be obtained by evaluating the resolution of two peaks as shown in Fig. 2–12.

Figure 2–12 Influence of the developing distance on the resolution of two peaks (for details, see text).

In Eq. 2.10 (adapted for TLC from Eq. 2.7), we can choose a selectivity of α= 1.5 and vary R_F in increments of 0.1. The plate number N is calculated from Eq. 2.3 as the quotient of developing distance (L) and the corresponding average plate height (H). Values for H are obtained from the graph for the HPTLC plate in normal development in Eq. 2.13 Regardless of R_F value, resolution

always shows a broad maximum for developing distances between 5 and 7 cm with a peak at 6 cm.

These calculations are proved experimentally in Fig. 2–14. When we compare the separation of the two compounds of chamomile oil marked with an arrow, it seems at first that it improves steadily with separation distance (Fig. 2–14A). With increasing distance of their maxima, also the thickness of the bands (widths of the corresponding peaks) grows. Normalizing all chromatograms reveals that resolution actually is the best at 6 cm developing distance (arrow, Fig. 2–14B). The same conclusion can be drawn from the analog curves of the chromatograms (Fig. 2–14C).

Similarly, the separation on TLC plates could be discussed. There, a developing distance of 12 to 15 cm gives best results.

Forced Flow

As long as the migration of the mobile phase depends on capillary forces, very little can be done to improve the efficiency of the TLC separation other than using smaller particles. Forcing the mobile phase through the layer with optimum velocity would change the situation dramatically. Low average plate heights could be maintained over long separation distances. The technical challenges of forced flow development have principally been solved in overpressured layer chromatography (OPLC), and numerous papers were published on the technique. For further details on the theory, see Ref. 11. Unfortunately, OPLC has still not found acceptance as a general separation technique.

The Influence of the Gas Phase

Another specialty of TLC is the presence of a gas phase in the developing chamber. This gas phase can interact with stationary and mobile phases and even with the sample. On one side, it is an advantage, because certain effects can be used to modify and improve a given separation. On the other side, it makes it nearly impossible to control and predict the outcome of a TLC separation based on mathematical models. The following sections approach some effects from a theoretical point of view and attempt to explain them. Practical consequences are described in “Chromatogram Development” in Chapter 3.

Processes in the Developing Chamber

In the following, we want to look at what happens in a “classical” developing chamber. As stationary phase we select silica gel and as mobile phase a mixture of toluene and ether (90:10, v/v). The development can be described as adsorption chromatography.

Four partially competing processes occur, as illustrated in Fig. 2–15.

The chamber represents a closed system in which the components of the developing solvent eventually establish phase equilibrium with their vapor. This equilibrium is called chamber saturation (1 in Fig. 2–15). Depending on the vapor pressure of the individual components, the composition of the gas phase can differ significantly from that of the developing solvent. In our example, we would expect the gas phase to contain much more ether (b.p. 35℃) than toluene (b.p. 110℃). We must also keep in mind that air (oxygen, nitrogen, and water vapor) contribute to the pressure in the tank. The time it takes to reach saturation depends on the ratio of solvent volume and surface to chamber volume and geometry. The process can be sped up if one or more walls of the chamber are lined with filter paper soaked with developing solvent.

The second process, called preconditioning (2 in Fig. 2–15), takes place when the dry stationary phase interacts with the solvent molecules present in the gas phase. In case the plate is not in contact with the developing solvent (as in a horizontal chamber; see Fig. 3–30C, page 101), eventually equilibrium would be reached. This equilibrium is called sorptive saturation: the surface of the stationary phase particles is covered by a thin layer of solvent molecules. The composition of that layer depends on the strength of adsorptive interaction. In our example, there would be more ether molecules absorbed than toluene molecules because the ether is more polar and can engage in stronger interaction with the silica gel. A consequence of sorptive saturation is the formation of a so-called virtual front below the upper “visible” front. The rising mobile phase “pushes” preadsorbed solvent molecules ahead and eventually creates a fully wet and thus visible area with a distinguished front at the top. This wet area is not accessible for migrating substances, and all R_F values turn out to be lower. Figure 2–16 and Box 2–1 illustrate this effect further.

Figure 2–16 The “virtual” front as result of preconditioning. Ethanol colored with Sudan blue. (A) Sandwich chambers. (B) Saturated chamber. For details, see Box 2–1.

Even if the development is performed without purposeful preconditioning, this effect of lowering the R_F values can be seen, particularly if the plate is developed over a long distance. This requires longer times and allows undesired preconditioning of the dry portions of the layer during the chromatography Fig. 2–14B.

During the plate development, the mobile phase rises through the layer, and unless the chamber is fully saturated (which in practice is rarely the case), part of the migrating liquid evaporates into the chamber. This evaporation (3 in Fig. 2–15) is controlled not only by the vapor pressures as in saturation (1) but also by the adsorption equilibriums (2) of the mobile phase components. When a plate is developed in an unsaturated chamber, evaporation becomes more important the higher the mobile phase rises. This effect slows down the migration of the mobile phase and can alter its composition.

While migrating, the components of the mobile phase may be separated (like sample components) by the stationary phase due to different degree of interaction. In our example, ether would be more strongly retained than toluene. As a result, formation of secondary fronts (4 in Fig. 2–15) can be observed. This effect is weaker the better the chamber is saturated and the more the stationary phase is preconditioned.

In most cases, TLC proceeds in a nonequilibrium between stationary, mobile, and gas phases. Reproducible chromatographic results can only be obtained if all external parameters are kept as constant as possible. Chamber form and chamber saturation play a dominant role in this regard. For an in-depth treatment of this and the following topic, please refer to Ref. 1.

Special Aspects of Sandwich Chambers

When the stationary phase is covered with a counter plate at less than 3 mm distance (but not in contact), a so-called sandwich configuration is obtained. In a sandwich chamber, preconditioning of the layer can be efficiently prevented. This generally gives rise to the formation of secondary fronts (Fig. 2–17), also called demixing. For each component of the mobile phase, a secondary front is formed. The R_F values of those fronts depend on the proportions of the corresponding solvents in the mobile phase. As far as the chromatographic result is concerned, it is often observed that more than one sample component migrates in secondary fronts resulting in zones without separation. Furthermore, such zones are often stretched sideways and have a very sharp upper edge. This makes quantitative evaluation difficult. Above the secondary front, the chromatographic result is similar to that obtained with a single solvent (the least polar of the mixture).

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