Stationary phase surface solute interaction with

Hydrophobic Interaction Chromatography. Hydrophobic interactions of solutes with a stationary phase result in thek adsorption on neutral or mildly hydrophobic stationary phases. The solutes are adsorbed at a high salt concentration, and then desorbed in order of increasing surface hydrophobicity, in a decreasing kosmotrope gradient. This characteristic follows the order of the lyotropic series for the anions ... 

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. 

Interactions of a Solute with a Stationary Phase Surface... 

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. 

The second type of interaction, displacement interaction, is depicted in Figure 10. This type of interaction occurs when a strongly polar solute, such as an alcohol, can interact directly with the strongly polar silanol group and displaces the adsorbed solvent layer. Depending on the strength of the interaction between the solute molecules and the silica gel, it may displace the more weakly adsorbed solvent and interact directly with the silica gel but interact with the other solvent layer by sorption. Alternatively, if solute-stationary phase interactions are sufficiently strong, then the solute may displace both solvents and interact directly with the stationary phase surface. 

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. 

Concentration of Chloroform in n-Heptane %w/v In contrast, the interactions with the stationary phase are becoming weaker as the surface becomes covered with chloroform. Thus retention is reduced by both the increased interactions in the mobile phase and reduced interaction with the stationary phase. When the concentration of chloroform in the solvent mixture is in excess of 50%, then the interactive properties of the stationary phase no longer change as the surface is now covered with a mono-layer of chloroform. However, solute retention will continue to decrease due to the increased interactions of the solute with the higher concentrations of chloroform in the mobile phase. It is clear that even with this simple example the dependence of retention on solvent composition is quite complex. 

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... 

Apart from enabling rapid prediction of solute retention, the Soczewinski equation allows a moleeular-level scrutiny of the solute — stationary phase interactions. The numeiieal value of the parameter n from Equation 2.14, which is at least approximately equal to unity (n 1), gives evidence of the one-point attachment of the solute moleeule to the stationary phase surface. The numeiieal values of n higher than unity prove that in a given chromatographic system, solute molecules interact with the stationary phase in more than one point (the so-ealled multipoint attachment). 

Two limiting mechanisms for solute retention can be imagined to occur in RPC binding to the stationary phase surface or partitioning into a liquid layer at the surface. In the previous treatment we assumed that retention is caused by eluite interaction with the hydrocarbonaceous surface, i.e., the first type of mechanism prevails. When the eluent is a mixed solvent, however, the less polar solvent component could accumulate near the apolar surface of the stationary phase. In the extreme case, an essentially stagnant layer of the mobile phase rich in the less polar solvent could exist at the surface. As a result eluites could partition between this layer and the bulk mobile phase without interacting directly with the stationary phase proper. 

For analysis of basic compounds, silica gel which has been sprayed with a solution of KOH in methanol, may be used. Treating the plate with base ensures that basic compounds chromatograph as their free bases rather than as their salts. The salts of the amines have very low mobility in organic solvent-based mobile phases since basic compounds tend to interact strongly with silanol groups on the surface of the silica the presence of KOH in the stationary phase suppresses this interaction. 

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 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,... 

The selectivity is primarily dependent on the nature of the analytes and their interaction with the stationary phase surface. If a dramatic change of the selectivity is needed for a particular separation, the best solution is the replacement of the type of the stationary phase. 

Inside the column, solutes are affected by the presence of micelles in the mobile phase and by the nature of the alkyl-bonded stationary phase, which is coated with monomers of surfactant (Fig. 1). As a consequence, at least two partition equilibria can affect the retention behavior. In the mobile phase, solutes can remain in the bulk water, be associated to the free surfactant monomers or micelle surface, be inserted into the micelle palisade layer, or penetrate into the micelle core. The surface of the surfactant-modified stationary phase is micelle-like and can give rise to similar interactions with the solutes, which are mainly hydrophobic in nature. With ionic surfactants, the charged heads of the surfactant in micelles and monomers adsorbed on the stationary phase are in contact with the polar solution, producing additional electrostatic interactions with charged solutes. Finally, the association of solutes with the nonmodified bonded stationary phase and free silanol groups still exists. 

---