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General classification of the modes of thin-layer chromatography (TLC) is based on the chemical nature of the stationary and mobile phases. The following three types of thin-layer chromatography given are widely recognized as different modes:
In this mode, active inorganic adsorbents (e.g., silica, alumina, or Florisil) are usually employed as stationary phases and, hence, the overall mechanism of retention is governed predominantly by the specific intermolecular interactions between the functionalities of the solutes, on the one hand, and active sites on the adsorbent surface, on the other. In adsorption TLC, aqueous mobile phases are never used, and stationary-phase activity prevails over the polarity of the mobile phase employed.
This mode of chromatography usually involves organic chemically bonded stationary phases with polar (e.g., 3-cyanopropyl) ligands.This particular mode is characterized by a mixed mechanism of solute retention: Solute molecules interact specifically with the polar functionalities of the organic ligand and with the residual active sites of the silica matrix, whereas their interactions with the hydrocarbon moiety of the organic ligands are entirely nonspecific in nature. Again, aqueous mobile phases are never employed in normal-phase (NP) TLC, and stationary phase activity prevails over the polarity of the mobile phase employed.
This chromatographic mode usually involves aliphatic chemically bonded stationary phases with, for example, octyl, octadecyl, or phenyl ligands. The mode of chromatography also is characterized by a mixed mechanism of solute retention: Solute molecules interact specifically with the residual active sites of the silica matrix, whereas their interactions with the aliphatic ligands are nonspecific (and predominantly hydrophobic) in nature. Reversed-phase (RP) TLC is usually performed with aqueous mobile phases containing organic modifiers [such as, e.g., methanol, acetonitrile (ACN), tetrahydrofuran (THF), etc.], and in this case, the activity of the stationary phase—as an exception—is less than that of the high-polarity mobile phase employed.
The parameter Rf is the quantity most commonly used to express the position of a solute in the developed chromatogram. It is calculated as a ratio:
Fig. 1
The thin-layer parameters used to
calculate the retention parameter Rf.
Using symbols from Fig. 1, Rf can be given as:
Rf values vary between 0 (solute remains at the origin) and 0.999 (solute migrates with the mobile-phase front). From a practical standpoint, the most reliable analytical results are achieved when the parameter Rf ranges from 0.20 to 0.80.
In the theory and practice of chromatography, another parameter of solute retention is also employed, the so-called RM value. This quantity was defined by Bate-Smith and Westall1 as
The adsorption and partition mechanisms of solute retention are the two most universal mechanisms of chromatographic separation, both operating on a physical principle. In fact, almost all solutes can be adsorbed on a microporous solid surface or be partitioned between two immiscible liquids. It is the main aim of semiempirical chromatographic models to couple the empirical parameters of retention with the established thermodynamic quantities generally used in physical chemistry. The validity of these models in chromatographic practice can hardly be overestimated, because when incorporated in separation selectivity-oriented optimization strategies, they often successfully help overcome the old trial-and-error approach used to optimize analyses. In the forthcoming sections, we will discuss a selection of the most popular and best-performing models and concepts of solute retention.
Partition chromatography was the first chromatographic technique to be given a thermodynamic foundation, by the pioneering work of Martin and Synge2, the 1952 Nobel Prize winners for chemistry. The Martin and Synge model describes the idealized parameter Rf (i.e., the parameter Rf) as
where tm and ts denote the time spent by a solute molecule in the mobile and stationary phases, respectively, nm and ns are numbers of solute molecules present in the mobile and stationary phases at equilibrium, and mm and ms are the respective numbers of moles.
Equation 4 can be further transformed as follows:
where cm and cs are the molar concentrations of the solute in the mobile and stationary phases, respectively, and Vm and Vs are volumes of these phases, respectively.
Assuming that
where K is the thermodynamic equilibrium constant for solute partitioning, we obtain
where is the so-called phase ratio (i.e., = Vs/Vm).
Equation 7 unites the empirical retention parameter of the solute, Rf, with the established thermodynamic (i.e., theoretical) quantity K, expressed as
where p0 is the standard chemical potential for partition. Hence, the retention model given by Eq. 8 can rightfully be called semiempirical.
The semiempirical model of adsorption chromatography, analogous to that of Martin and Synge, was established only in the late 1960s by Snyder3 and Soczewinski4 independently, and it is often referred to as the displacement model of solute retention. The crucial assumption of this model is that the mechanism of retention consists in competition among the solute and solvent molecules for the active sites of the adsorbent and, hence, in a virtually endless process of the solvent molecules displacing those of the solute from the solid surface (and vice versa). Further, the authors assumed that some of the mobile phase remains adsorbed and stagnant on an adsorbent surface.
This adsorbed mobile phase formally resembles the liquid stationary phase in partition chromatography. Thus—utilizing with imagination the main concept of the Martin and Synge model of partition chromatography—Snyder and Soczewinski managed to define the Rf parameter valid for adsorption chromatography as
where tm and ta denote the time spent by a solute molecule in the mobile phase and on the adsorbent surface, respectively, nm and na are numbers of solute molecules contained in the mobile phase and on the adsorbent surface at equilibrium, respectively, mm and ma are the numbers of moles of solute molecules contained in the nonadsorbed and adsorbed moieties of the mobile phase, respectively, cm and ca are molar concentrations of solute in the nonadsorbed and the adsorbed moieties of mobile phase, respectively, Vm is the total volume of mobile phase, Va is the volume of the adsorbed mobile phase per unit mass of adsorbent, and Wa is the mass of adsorbent considered. Transformation of Eq. 9 results in the relationship
where Kth = ca/cm, Kth being the thermodynamic equilibrium constant of adsorption, and = VaWa/(Vm - VaWa).
Two very simple relationships have been derived from the general framework of the Snyder and Soczewinski model of adsorption chromatography; these have proved useful for rapid prediction of solute retention in chromatographic systems employing binary mobile phases. One (known as the Soczewinski equation) proved successful for adsorption and normal-phase TLC; the other (known as the Snyder equation) proved similarly successful in reversedphase TLC. Soczewinski Equation.
The Soczewinski equation (5) [Eq. 11 ] is a simple linear relationship with respect to log Xs, linking the retention parameter (i.e., Rm) of a given solute with the quantitative composition of the binary mobile phase used:
where C is, in the first instance, the equation constant (although with clear physicochemical significance), Xs is the molar fraction of the stronger solvent in the nonaqueous mobile phase, and n is the number of active sites on the surface of the adsorbent.
Apart from enabling rapid prediction of solute retention, the Soczewinski equation enables molecularlevel scrutiny of solute–stationary phase interactions. Thus, a numeric value of the parameter n of Eq. 11 of approximately unity (n 1) implies one-point attachment of the solute molecule to the stationary-phase surface. Numerical values of n higher than unity indicate that in a given chromatographic system, solute molecules interact with the stationary phase at more than one point (so-called multipoint attachment).
The Snyder equation6 [Eq. 12 ] is another simple linear relationship with respect to , which links the retention parameter (i.e., ln k) of a given solute with the volume fraction of the organic modifier in the aqueous binary mobile phase ():
where k is the retention coefficient of the solute [k=(1-Rf)/Rf], kw is the retention coefficient extrapolated for pure water as the mobile phase, and S is a constant characteristic of a given stationary phase.
Consequences of the Snyder and Soczewinski model are manifold, and they are of significant practical importance. The most spectacular conclusions of this model are (a) the possibility of quantifying the activity of an adsorbent and (b) the possibility of defining and quantifying the "chromatographic polarity" of solvents (known as their elution strength). These two conclusions could be drawn only upon the assumption of the displacement mechanism of solute retention. An obvious necessity in this model was to quantify the effect of displacement, which resulted in the relationship given by Eq. 13 for the thermodynamic equilibrium constant of adsorption, Kth, for an active chromatographic adsorbent and a monocomponent mobile phase:
where @alpha is a function of the adsorbent surface energy and is independent of the properties of the solute (it is known as the activity coefficient of the adsorbent; practical determination of its numerical values can be regarded as quantification of adsorbent activity), S0 is the adsorption energy of a solute chromatographed on an active adsorbent with n-pentane as the mono-component mobile phase, As is the surface area of the adsorbent occupied by an adsorbed solute molecule, and ε0 is the parameter usually referred to as the solvent elution strength, or simply solvent strength (it is the energy of adsorption of solvent per unit surface area of adsorbent).
Assuming that the adsorbent surface occupied by an adsorbed solute molecule (AS) and that occupied by a stronger solvent (nB) are equal, the eluent strength of a binary mobile phase, AB has the following dependence on its quantitative composition:
where εA is the eluent strength of the weaker component (A) of a given binary mobile phase, B is the eluent strength of the stronger component (B) of the same mobile phase, and xB is the molar volume of the component B.
Combining Eqs. 13 and 14 gives the following relationship, which expresses the dependence of the retention parameter of a solute, RM (= log k), on the quantitative composition of a given binary mobile phase:
In this particular model, it is assumed that Hildebrand's concept of the solubility parameter (δ), originally formulated for liquid nonideal solutions, can also be applied to the solute and to the stationary and mobile phases of chromatographic systems.
The solubility parameter of any given substance (δ) is defined as
where E denotes its heat of vaporization at zero pressure, -E is the energy of cohesion needed for transportation of one mole of an ideal gas phase to liquid phase, and V is the molar volume of the liquid.
One of the basic retention parameters (i.e., the solute's retention coefficient k) can be expressed as a function of the solubility parameters, δ:
where vi is the molar volume of the ith solute, δi, δs and δm are respectively the solubility parameters of this solute and of the stationary and mobile phases employed, and ns and nm are respectively the numbers of moles of the stationary and mobile phases. Finally, the principal equation of the Schoenmakers model10 of solute retention in reversed-phase chromatography employing a binary aqueous mobile phase takes the parabolic form:
where the equation constants A, B, and C have a clear physicochemical significance:
where δw and δa denote respectively the solubility parameters of water and of the organic modifier as the constituents of a given aqueous mobile phase.