Bonded stationary phases reversed phase retention

Jinno, K., Effect of the aUcyl chain length of the bonded stationary-phase on solute retention in reversed phase high performance hquid chromatography, Chromato-graphia, 15, 667, 1982. 

Variations in retention and selectivity have been studied in cyano, phenyl, and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline, and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile, or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns were correlated with polar group selectivities of mobile-phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers [84],... 

Nahum, A. and Horvath, C. (1981) Surface silanols in silica-bonded hydrocarbonaceous stationary phases. I. Dual retention mechanism in reversed-phase chromatography. J. Chromatogr. 203, 53 63. 

Polar-bonded stationary phases, such as cyanopropyl, diol, or aminopropyl, bonded to a silica matrix, have moderate polarity and can be used in normal- and reversed-phase (RP) systems. The retention behavior of heterocyclic bases was also examined using these adsorbents by determination of Rm (log k) values of solutes by the use of eluents with various modifier concentrations.It was statistically found that the Snyder-Soczewihski equation and Scott theory describe the retention of quinolines on polar-bonded stationary phases in normal-phase systems sufficiently well. It seems that results are consistent with a displacement model. The dispersive interactions between solute molecules and the polar component of an eluent seem also to have an important role. Similarly, the retention—... 

Alhedai et al also examined the exclusion properties of a reversed phase material The stationary phase chosen was a Cg hydrocarbon bonded to the silica, and the mobile phase chosen was 2-octane. As the solutes, solvent and stationary phase were all dispersive (hydrophobic in character) and both the stationary phase and the mobile phase contained Cg interacting moieties, the solute would experience the same interactions in both phases. Thus, any differential retention would be solely due to exclusion and not due to molecular interactions. This could be confirmed by carrying out the experiments at two different temperatures. If any interactive mechanism was present that caused retention, then different retention volumes would be obtained for the same solute at different temperatures. Solutes ranging from n-hexane to n hexatriacontane were chromatographed at 30°C and 50°C respectively. The results obtained are shown in Figure 8. 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Buszewski, B., Gadza-la-Kopciuch, R. M., Markuszewski, M. L, Kaliszan, R. Chemically bonded silica stationary phases synthesis, physicochemical characterization, and molecular mechanism of reversed-phase HPLC retention. Anal. Chem. 1997, 69, 3277-3284. 

Several improved stationary phase materials have been synthesized for reversed-phase liquid chromatography. One material is vinyl alcohol copolymer gel. This stationary phase is quite polar and chemically very stable however, it demonstrated a strong retention capacity for polycyclic aromatic hydrocarbons.45 9 Although stable octadecyl- and octyl-bonded silica gels have been synthesized from pure silica gel50,51 and are now commercially available, such an optimization system has not yet been built. Further experiments are required to elucidate the retention mechanism, and to systematize it within the context of instrumentation. 

Reversed-phase chromatography employs a nonpolar stationary phase and a polar aqueous-organic mobile phase. The stationary phase may be a nonpolar ligand, such as an alkyl hydrocarbon, bonded to a support matrix such as microparticulate silica, or it may be a microparticulate polymeric resin such as cross-linked polystyrene-divinylbenzene. The mobile phase is typically a binary mixture of a weak solvent, such as water or an aqueous buffer, and a strong solvent such as acetonitrile or a short-chain alcohol. Retention is modulated by changing the relative proportion of the weak and strong solvents. Additives may be incorporated into the mobile phase to modulate chromatographic selectivity, to suppress undesirable interactions of the analyte with the matrix, or to promote analyte solubility or stability. 

Another approach to preparing a stable reversed phase with fewer residual silanols is the use of polyfunctional silanes of the type R2SiX2. These react to form a polymeric stationary phase that shields the siloxane bonds and restricts access to residual silanols. Polymer phases have higher carbon loads and are typically more retentive than monomeric phases. However, they are more difficult to synthesize reproducibly and may exhibit batch-to-batch variability in their properties. They also exhibit poorer mass transfer kinetics and so provide poorer efficiency than monomeric phases. 

Reversed-phase liquid chromatography shape-recognition processes are distinctly limited to describe the enhanced separation of geometric isomers or structurally related compounds that result primarily from the differences between molecular shapes rather than from additional interactions within the stationary-phase and/or silica support. For example, residual silanol activity of the base silica on nonend-capped polymeric Cis phases was found to enhance the separation of the polar carotenoids lutein and zeaxanthin [29]. In contrast, the separations of both the nonpolar carotenoid probes (a- and P-carotene and lycopene) and the SRM 869 column test mixture on endcapped and nonendcapped polymeric Cig phases exhibited no appreciable difference in retention. The nonpolar probes are subject to shape-selective interactions with the alkyl component of the stationary-phase (irrespective of endcapping), whereas the polar carotenoids containing hydroxyl moieties are subject to an additional level of retentive interactions via H-bonding with the surface silanols. Therefore, a direct comparison between the retention behavior of nonpolar and polar carotenoid solutes of similar shape and size that vary by the addition of polar substituents (e.g., dl-trans P-carotene vs. dll-trans P-cryptoxanthin) may not always be appropriate in the context of shape selectivity. 

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. 

Eluents used in reversed-phase chromatography with bonded nonpolar stationary phases are genei ly polar solvents or mixtures) of polar solvents, such as acetonitrile, with water. The properties of numerous neat solvents of interest, their sources, and their virtues in teversed-phase chromatography have been reviewed (128). Properties of pure solvents which may be of value as eluents are summiuized in Table. VII. The most significant properties are surface tension, dielectric constant, viscosity, and eluotropic value. Horvath e/ al. 107) adapted a theory of solvent effects to consider the role of the mobile phase in determinmg the absolute retention and the selectivity found in reversed-phase chromatography. 

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