Reversed-phase selectivity characterization

Lopez, L. A. and Rutan, S. R., Comparison of Methods for Characterization of Reversed-phase Liquid Chromatograpic Selectivity, /. Chromatogr. A, 965 301-314, 2002. 

While much has been learned about the role and selection of the operation parameters in displacement chrcmatography (60-66), little is known yet about the rules of displacer selection and the means available to control the selectivity of the separation. The paucity of well characterized displacers and the lack of knowledge of the solute adsorption isotherms hinder most strongly the wider acceptance and use of displacement chromatography. In most cases, displacer selection is still done by trial-and-error. In the majority of modem displacement chromatographic publications a reversed-phase system was used to separate small polar molecules, antibiotics, oligopeptides and small proteins (52-66). 

The previous chapter discussed the solvent and its interaction with the solute. To complete the chromatographic system the adsorbent has to be selected. As mentioned in Chapter 3.2.1 one has to distinguish between enantioselective and non-enantioselective adsorbents. Both groups of adsorbents are classified into polar, semi-polar and nonpolar adsorbents (see Tab. 4.4). This classification is based on the surface chemistry of the packing material. Interaction between mobile phase and adsorbent characterizes the phase system, which is distinguished between normal phase (NP) chromatography and reversed phase (RP) chromatography. This differentiation is historic and appointed by the ratio of the polarity of the adsorbent and the mobile phase. 

A unified theory recently proposed to explain the manner of sorption and the form of sorption isotherm in gas, liquid, and ion-exchange chromatography is presented in some detail. Selectivity in reversed-phase high-pressure liquid chromatography is explored at length. Several chapters deal with characterization of bonded phases, relationship of column-packing structure and performance, variability of reversed-phase packing materials, and the differences between silica-based reversed-phase and poly(styrene-divinylbenzene) columns. A short review is included to cover various approaches used in HPLC to achieve the desired selectivity for resolution of enantiomeric compounds. 

Nevertheless, the best overall characterization of a reversed-phase bonded phase is done by well-designed chromatographic tests. Such a test should enable us to chai erize at least the hydrophobidty (retentivity) of a packing and the activity of the residual silanols. As we will see, tests can also be designed to characterize the shape selectivity ot a packing or its behavior toward complexing agents. 

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