Reversed-phase chromatography solute retention mechanisms

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

Sentell, K.B. and Dorsey, J.G, Retention mechanisms in reversed-phase chromatography. Stationary-phase bonding density and solute selectivity, J. Chromatogr., 461, 193, 1989. 

Although the detailed retention mechanisms are as yet unclear (see for example 7.8). there is a building consensus that reversed phase chromatography is dominated by the hydrophobic effect. Retention is therefore primarily a function of solution phenomena in the mobile phase, and it is not surprising that RPLC has many ways to modify selectivity by manipulating the chemical nature of the mobile phase. 

Virtually all interactive mechanisms that control retention in chromatography are, in fact, mixed interactions as shown by the previous application examples. It has already been suggested that reverse phases can exhibit almost exclusively dispersive interactions with solutes. However, as they are almost always employed with aqueous solvent mixtures then, polar and dispersive interactions will still be operative in the mobile phase. Consequently, the examples given here will be taken where the mixed interactions are either unique or represent a separation of special interest. 

Dill, K.A., The mechanism of solute retention in reversed-phase liquid chromatography, J. Phys. Chem., 91, 1980, 1987. 

The exact mechanism(s) of solute retention in reversed-phase high-performance liquid chromatography (RPLC) is not presently well understood. The lack of a clear understanding of the mechanics of solute retention has led to a myriad of proposals, including the following partition (K21, L6, S16) adsorption (C9, CIO, H3, H15, H16, K13, L3, T2, U2) dispersive interaction (K2) solubility in the mobile phase (L7) solvophobic effects (H26, K6, M5) combined solvophobic and silanophilic interaction (B9, M12, Nl) and a mechanism based upon compulsary absorption (B5). 

Micelles and cyclodextrins are the most common reagents used for this technique. Micellar electrokinetic capillary chromatography (MECC or MEKC) is generally used for the separation of small molecules [6], Sodium dodecyl sulfate at concentrations from 20 to 150 mM in conjunction with 20 mM borate buffer (pH 9.3) or phosphate buffer (pH 7.0) represent the most common operating conditions. The mechanism of separation is related to reversed-phase liquid chromatography, at least for neutral solutes. Organic solvents such as 5-20% methanol or acetonitrile are useful to modify selectivity when there is too much retention in the system. Alternative surfactants such as bile salts (sodium cholate), cationic surfactants (cetyltrimethy-lammonium bromide), nonionic surfactants (poly-oxyethylene-23-lauryl ether), and alkyl glucosides can be used as well. 

The adsorption from solutions of polycyclic aromatic hydrocarbons, alkylbenzenes and benzene derivatives is of great interest from many points of view. To understand the mechanism of their adsorption and to predict the equilibrium adsorption constant also it is possible to use the contributions of functional groups of fragments of molecules to retention in reversed-phase and normal-phase liquid chromatography [21]. The contributions of different molecular groups as well as the dependence of these contributions on mobile phase composition have been evaluated from experimental data in work [25]. 

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