Reversed-phase chromatography test mixture

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. 

In a similar way, Figure 6.65 shows the comparison of ion-pair and mixedmode seiectivities for the separation of phenylphosphonate and aromatic sulfonic acids. While all components of the test mixture can be separated to baseline on a mixed-mode phase, the separation of di- and trisulfonic acids with ion-pair chromatography is not possible at all. Both examples clearly demonstrate that for an optimal separation of ionic and strongly polar compounds, the combination of ion-exchange and reversed-phase interactions is of great importance. 

Figure 2 Reversed-phase high-performance liquid chromatography (RP-HPLC) of a test mixture composed of amino acids and hydrophilic peptides.

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