Download (direct link):
In reversed-phase HPLC, when a nonpolar hydrocarbon stationary phase and a hydroor-ganic mobile phase are applied, the retention of the solute is governed mostly by hydrophobic forces (the strength of the solute-stationary-phase interaction is determined by the hydropho-bicitv of the compounds).
In adsorption chromatography the strength of the solute-stationary-phase interaction is determined by adsorption forces. The adsorption interaction is the result of various types of forces, such as dispersive, inductive, or hydrogen-bond formation.
In ion-exchange chromatography the stationary phase has an ion-exchanger group, which interacts with the solute molecules, and the interaction is modulated by the presence of other displacing ions in the mobile phase.
In gel-filtration chromatography the separation takes place according to the size of the molecules. The smaller molecules elute later from the column, because they spent more time
in the pores of the stationary phase. Gel-filtration chromatography can be applied to determine
the size of the molecules. The method can be applied only for large molecules (over 1000
In hydrophobic-interaction chromatography, which is mostly applied in the analytical or preparative separation of biomolecules, the solute-stationary-phase interaction (which is ba-sically hydrophobic, as in reversed-phase) is modulated by the gradually decreasing concen-tration of salt molecules in the mobile phase.
In chiral chromatography, the mechanism of the solute-stationary-phase interaction can be of various types, but it should be enantioselective. Usually, at least three interacting points between the solute and the stationary phase should be present close to the chiral center of the molecule. These interactions can be hydrophobic, adsorptive, H-bonding, steric, or tt-tt interactions.
It can be concluded that the stationary phases can be designed to interact with the solute molecules by various mechanisms. In practice the stationary phases are designed for certain separation problems. What can a chemist do when he or she wants to use chromatography for physicochemical measurements? On the basis of the different mechanisms of solute-stationary-phase interaction, he or she can select among the commercially available stationary phases, or it is possible to apply various mobile-phase additives, by which a so-called dynamically coated stationary phase can be prepared. The peak shape and the efficiency of the column should always be checked. An imprecise peak shape can reveal secondary equilibria or multiple retention mechanisms. When we use the retention data for the determination of molecular interactions or physicochemical measurements we always have to know the type of interactions and the retention mechanism to be able to draw valid conclusions. The retention will always be the result of a combination of all the solute-stationary phase interactions.
Reversed-phase chromatography provides the simplest way to determine solute-stationary-phase interactions that will indicate the hydrophobic character of the solute. Commercially available reversed-phase columns provide good efficiency, excellent reproducibility, and a long lifetime for accurate retention determination. Therefore, the most widespread application of HPLC for physicochemical measurements is the determination of partition coef-ficients; in other words, the hydrophobic properties of compounds. Depending on the chemical-bonding technology and the amount of coating, reversed-phase columns can be con-sidered as inert phases, and only the hydrophobic interactions govern the retention, although in special cases, the presence of the free silanol groups might cause a dual retention mecha-nism. The methods for the application of RP-HPLC for hydrophobicity determination of com-pounds will be discussed in detail. The application of HPLC for adsorption isotherms, mo-lecular weight, shape and size, and molecular interactions will be briefly summarized.
II. DETERMINATION OF HYDROPHOBICITY BY HPLC
A. The Role of Hydrophobicity in Biomedical Sciences
Overton  and Meyer  first recognized the role of hydrophobicity of compounds in their pharmacological activity. They described a correlation between the narcotic effect and the partition of compounds between an aqueous and a lipid layer. Since then, several thousand papers have discussed the correlations between the hydrophobicity of the compounds and their pharmacological activity. The absorption of compounds from soil by plants ; the distribution of drug molecules in the body, and their probability of reaching the active site of a receptor ; or the bioaccumulation of the chemicals in soil, natural waters, fish  and so on, depend on the hydrophobic nature of the compound. These correlations are based on the fact that both the soil and the living systems contain hydrophobic and hydrophilic compartments, among which the molecules should be partitioned. In drug-receptor interactions or enzyme-substrate interactions, hydrophobic forces may play an important role, which can be