Solute interaction with stationary phase

Figure 1 Solute interactions with stationary phase and micelle in pure MLC systems containing the surfactants (A) SDS and (B) CTAB,...

It must be emphasized that a mixture containing micelles will not necessarily produce a simple, single component stationary (or non aqueous) phase. Not only will the solute be distributed between the micelles and the aqueous phase, but also, to a lesser or greater extent, will any other component in the electrophoretic solution. The presence of any other organic component distributed between the micelles and the aqueous phase will modify the solute interactions with both phases. In general, increased interaction of the solute with the micelles will slow the migration rate of the solute. Increased interaction of the solute with the aqueous phase will increase the migration rate of the solute. 

In the most common application of this separation mode, components are separated according to the number and nature of the polar functional groups (e.g., ester bonds, phosphate, hydroxyl, and amine groups) in lipid molecules. Since the head group of an individual lipid class predominantly determines the polar interactions with stationary phase, normal-phase HPLC separates a lipid extract solution into the lipid classes rather than into molecular species. 

In an attempt to explain the nature of polar interactions, Martire et al. [15] developed a theory assuming that such interactions could be explained by the formation of a complex between the solute and the stationary phase with its own equilibrium constant. Martire and Riedl adopted a procedure used by Danger et al. [16], and divided the solute activity coefficient into two components. 

When the silica surface is in contact with a solvent, the surface is covered with a layer of the solvent molecules. If the mobile phase consists of a mixture of solvents, the solvents compete for the surface and it is partly covered by one solvent and partly by the other. Thus, any solute interacting with the stationary phase may well be presented with two, quite different types of surface with which to interact. The probability that a solute molecule will interact with one particular type of surface will be statistically controlled by the proportion of the total surface area that is covered by that particular solvent. 

Retention is controlled by solute interactions with both the mobile phase and the stationary phase and each will be discussed in this chapter. Interactions in the mobile... 

There are two ways a solute can interact with a stationary phase surface. The solute molecule can interact with the adsorbed solvent layer and rest on the top of it. This is called sorption interaction and occurs when the molecular forces between the solute and the stationary phase are relatively weak compared with the forces between the solvent molecules and the stationary phase. The second type is where the solute molecules displace the solvent molecules from the surface and interact directly with the stationary phase itself. This is called displacement interaction and occurs when the interactive forces between the solute molecules and the stationary phase surface are much stronger than those between the solvent molecules and the stationary phase surface. An example of sorption interaction is shown in Figure 9. 

Where there are multi-layers of solvent, the most polar is the solvent that interacts directly with the silica surface and, consequently, constitutes part of the first layer the second solvent covering the remainder of the surface. Depending on the concentration of the polar solvent, the next layer may be a second layer of the same polar solvent as in the case of ethyl acetate. If, however, the quantity of polar solvent is limited, then the second layer might consist of the less polar component of the solvent mixture. If the mobile phase consists of a ternary mixture of solvents, then the nature of the surface and the solute interactions with the surface can become very complex indeed. In general, the stronger the forces between the solute and the stationary phase itself, the more likely it is to interact by displacement even to the extent of displacing both layers of solvent (one of the alternative processes that is not depicted in Figure 11). Solutes that exhibit weaker forces with the stationary phase are more likely to interact with the surface by sorption. 

Now, if the solutes interact with themselves more strongly than they do with the stationary phase, then their presence will increase the interaction of further solute with the stationary phase mixture. This gives an isotherm having the shape shown in Figure 10. This type of isotherm is called a Freundlich isotherm, the expression for... 

However, in LC solutes are partitioned according to a more complicated balance among various attractive forces solutes interact with both mobile-phase molecules and stationary-phase molecules (or stationary-phase pendant groups), the stationary-phase interacts with mobile-phase molecules, parts of the stationary phase may interact with each other, and mobile-phase molecules interact with each other. Cavity formation in the mobile phase, overcoming the attractive forces of the mobile-phase molecules for each other, is an important consideration in LC but not in GC. Therefore, even though LC and GC share a considerable amount of basic theory, the mechanisms are very different on a molecular level. This translates into conditions that are very different on a practical level so different, in fact, that separate instruments are required in modern practice. 

The types of interaction that can occur between the solute and the stationary phase surface when in contact with the pure solvents, n-heptane, chloroform or a mixture of n-heptane/chloroform are shown in figure 2. 

The possible alternatives for a solute molecule to interact with a bilayer of solvent molecules is depicted in figure 5. It is seen that such a surface offers a wide range of sorption and displacement processes that can take place between the solute and the stationary phase surface. There are, in fact, three different surfaces on which a molecule can... 

It is seen that to identify the impurities, the column appeared to be significantly overloaded. Nevertheless, the impurities were well separated from the main component and the presence of a substance was demonstrated in the generic formulation that was not present in the Darvocet . The mobile phase was 98.5% dichloromethane with 1.5% v/v of methanol containing 3.3% ammonium hydroxide. The ammoniacal methanol deactivated the silica gel but the interaction of the solutes with the stationary phase would still be polar in nature. In contrast solute interactions with the methylene dichloride would be exclusively dispersive. 

The relative distribution of a solute between two phases is determined by the interactions of the solute species with each phase. The relative strengths of these interactions are determined by the variety and the strengths of the intermolecular and other forces that are present, or, in more general terms by the polarity of the sample and that of the mobile and stationary phases. 

The thermodynamics of solute interaction with nonpolar ligates of the stationary phase will be treated later in this chapter within the framework of the solvophobic theory 107-108). According to this theoretical approach the equilibrium constant for the reversible binding of a given eluite to the hydrocarbonaceous ligates at fixed eluent properties and temperature can be approximated by the relationship... 

Polar interactions between molecules arise from permanent or Induced dipoles existing in the molecules and do not result from permanent charges as in the case of Ionic interactions. Examples of polar substances having permanent dipoles would be alcohols, ketones, aldehydes etc. Examples of polarizable substances would be aromatic hydrocarbons such as benzene or toluene. It is considered that, when a molecule carrying a permanent dipole comes Into close proximity to a polarizable molecule, the field from the molecule with the permanent dipole induces a dipole in the polarizable molecule and thus electrical interaction can occur. It follows that to selectively retain a polar solute, then the stationary phase must also be polar and contain, perhaps, hydroxyl groups. If the solutes to be separated are strongly polar, then perhaps a polarizable substance such as an aromatic hydrocarbon could be employed as the stationary phase. However, to maintain strong polar interactions with the stationary phase (as opposed to the mobile phase) the mobile phase must be relatively non-polar or dispersive in nature. 

The two functions involving either K2 or K3, or both, in equation (13) can, in theory, contribute to solute retention. This will depend on whether the solute interacts with the absorbed component of the mobile phase on the surface or penetrates the layer and interacts with the stationary phase proper. In either case, the accessibility of the solute to the retentive phase is governed by the magnitude of (n) and (fc) Another important aspect of equation (13) Is its Implication on the accuracy with which the capacity factor k can be measured. Now, (k ), is normally calculated in the following way,... 

The effect of temperature, pressure and density on solute retention (k1) in supercritical fluid chromatography (SFC) has been well studied.(1-6) Retention in SFC depends upon both solute solubility in the fluid and solute interaction with the stationary phase. The functional relationship between retention and pressure at constant temperature has been described by Van Wasen and Schneider. ( 1 ) The trend in retention is seen to depend on the partial molar volume of... 

The first fraction of solute emerging from a gel column is eluted in v() that contains molecules too large to enter the gel micropores. v0 is the macroscopic pore space in the gel bed, not otherwise participate in the sieving mechanism. For a given column, v0 is constant. The solute interacting with the liquid stationary phase on the column surface elutes in order of the magnitude of a fraction s partition coefficient (Kp) between the elution volume (vel) and the volume of stationary solvent in the micropores (vs), fixed at 100 mL, because of difficulty in its measurement (Bio-Rad, 1971). At Ts = 100 mL,... 

Snyder [350] has given an early description and interpretation of the behaviour of LSC systems. He explained retention on the basis of the so-called competition model . In this model it is assumed that the solid surface is covered with mobile phase molecules and that solute molecules will have to compete with the molecules in this adsorbed layer to (temporarily) occupy an adsorption site. Solvents which show a strong adsorption to the surface are hard to displace and hence are strong solvents , which give rise to low retention times. On the other hand, solvents that show weak interactions with the stationary surface can easily be replaced and act as weak solvents . Clearly, it is the difference between the affinity of the mobile phase and that of the solute for the stationary phase that determines retention in LSC according to the competition model. 

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